(Received for publication, May 10, 1995; and in revised form, July 14, 1995)
From the
The pre-myosin light chain (MLC) phosphorylation
components of the lag phase (t
) of
contractile activation were determined in permeabilized smooth muscles
activated by photolytic release of ATP from caged ATP and/or
Ca
from 4-(2-nitrophenyl)-EGTA (NP-EGTA). Calmodulin
(CaM) shortened the t
(470 ms at 0 added
CaM) that followed Ca
release, but its effect (t
=
200 ms) saturated at 40
µM. Photolysis of caged ATP following preequilibration
with identical [Ca
CaM] shortened t
to 41 ms. The rate of phosphorylation
was very fast (3.5 s
at 22 °C in the presence of
5 µM exogenous CaM) following photolysis of caged ATP,
and, following Ca
release, phosphorylation was
accelerated by CaM. Simultaneous photolysis of caged ATP and NP-EGTA
was followed by a t
of 194 ms at 5
µM CaM and a rate of MLC
phosphorylation
intermediate between these parameters following photolysis of,
respectively, NP-EGTA and caged ATP. In the presence of the normal,
total endogenous CaM content (37 ± 4 µM) of portal
vein smooth muscles t
was 565 ms.
Steady state maximum force at pCa 5.5 was increased by much
lower (100 nM) exogenous [CaM] than was required
(>2.5 µM) to shorten the t. We estimate the endogenous CaM
available under steady state conditions in vivo to be
approximately 0.25 µM and probably less during a rapid
Ca
transient.
We conclude that the [CaM]
dependence of the kinetics of MLC phosphorylation and
force development (t
and t
) initiated by Ca
reflects the recruitment of a slowly diffusible component of
total CaM. The relatively long duration of t
(197 ms) at saturating [CaM] suggests the
contribution to t
of an additional
component, possibly a prephosphorylation activation/isomerization of
the Ca
CaM myosin light chain kinase complex
(Török, K., and Trentham, D.
R.(1994) Biochemistry 33, 12807-12820). The relatively
short delay (108 ms in the presence of 40 µM CaM)
following simultaneous photolysis of NP-EGTA and caged ATP suggests
that preincubation with ATP (prior to photolysis of NP-EGTA) may
inhibit the formation of a preactive Ca
CaM myosin light
chain kinase complex.
A long lag phase between the binding of an excitatory agonist to
its receptor and contraction is a characteristic property of smooth
muscle, evident even when diffusional delays are eliminated through
photolysis of caged agonists (Somlyo et al., 1988a;
Muralidharan et al., 1993; Walker et al., 1993). A
major component of the delay (1-2 s in guinea pig portal vein
smooth muscle stimulated with phenylephrine at room temperature; Somlyo et al. (1988a)) is due to phospholipase C-mediated generation
of inositol 1,4,5-trisphosphate that is the messenger of
pharmacomechanical Ca release from the sarcoplasmic
reticulum (reviewed by Somlyo and Somlyo(1990)). Of the remaining
delay, only a small fraction (
30 ms; Somlyo et al., 1992)
is due to the time elapsed between the rise of inositol
1,4,5-trisphosphate and Ca
release; its major
component occurs after the rise in cytoplasmic Ca
,
whether the latter is the result of inositol
1,4,5-trisphosphate-induced Ca
release, electrical
stimulation, or Ca
spikes during spontaneous
electrical activity (Somlyo et al., 1988b, 1992; Yagi et
al., 1988; Himpens and Somlyo, 1988). We had suggested (Somlyo et al., 1988b) that this lag, which is much longer than the
few ms delay between the rise in [Ca
]
and contraction in striated muscle (Ellis-Davies and
Kaplan(1994); reviewed by Ashley et al. (1991)), could be the
result of prephosphorylation reactions and/or the time course of
phosphorylation of myosin light chains that activates contraction in
smooth muscle. The purpose of the present study was to determine the
relative contributions of these two components to the kinetics of
activation in smooth muscle. To rapidly and synchronously activate
contraction with Ca
, we used laser flash photolysis
of a new caged Ca
(NP-EGTA) (
)that
combines the advantages of a 4-order of magnitude decrease in affinity
for Ca
(K
80 nMversus 1 mM) upon photolysis with a low affinity
for Mg
(K
= 9
mM; Ellis-Davies and Kaplan(1994)). Varying the calmodulin
(CaM) concentration in permeabilized smooth muscle activated by
photolysis of the NP-EGTA-Ca
complex allowed us to
obtain a precise measure of both the delay and its dependence on
calmodulin concentration and to relate, through rapid freezing, the
mechanical to the biochemical events of MLC
phosphorylation. Comparison of the kinetics of contraction and
MLC
phosphorylation initiated through photolysis of,
respectively, NP-EGTA or caged ATP (the latter in smooth muscles
preequilibrated with Ca
-calmodulin) provided strong
evidence of the contribution of one or more significant kinetic steps,
attributable to prephosphorylation reactions, to the delay during the in vivo activation of smooth muscle by Ca
. A
preliminary report of some of these findings has been published
(Zimmermann et al., 1995).
The free Ca concentrations in these as well as in the solutions with various
[Ca
]
, constructed from
``G10'' and ``pCa 4.5,'' were measured
with Ca
-sensitive minielectrodes. Electrodes were
calibrated with EGTA-buffered calibration solutions (calcium
calibration kit with magnesium I; Molecular Probes Inc., Eugene, OR).
After recording contractions
evoked by depolarization with a high K solution, the
strips were permeabilized with 50 µg/ml
-escin in G0 (pCa 6) for 30-45 min and subsequently incubated for 10
min with 10 µM A23187 (Fig. 1). Photorelease of the
caged compounds was induced by a 50-ns flash of 347-nm light from a
frequency-doubled ruby laser (Lumonics Ltd., Warwickshire, United
Kingdom) focused to illuminate the entire muscle strip. All photolysis
experiments were carried out at room temperature (22-23 °C).
The system for data collection in the majority of the experiments was
identical to that described by Nishiye et al.(1993). Later
experiments were performed using an AT-M10-16X AD conversion card
at an acquisition rate of 500 Hz in combination with the data
acquisition software Labview 3.0 (National Instruments) on a PC. Force
transients were fitted using the software SigmaPlot (Jandel
Scientific).
Figure 1:
Force
records illustrating the experimental protocol used for activation by
photolysis of NP-EGTA and caged ATP. A, after the control
contraction elicited by depolarization with 143 mM K, the strips were permeabilized with
-escin, incubated for 10 min in A23187, washed in G0, and relaxed
for 12 min in photolysis solution, followed by photolysis of NP-EGTA
with a single laser pulse (arrow). B, the sequence of
incubations for the photolysis of caged ATP consisted of
permeabilization and incubation in A23187 (not shown), relaxation of
the strips in G10, removal of ATP in G10R, and an increase of
[Ca
]
in pCa 5.5R.
The preparations were finally incubated for 3 min in caged ATP
containing photolysis solution and activated by photolytic liberation
of 2.7 ± 0.2 mM ATP (arrow).
All solutions were continuously pumped through the capillary until shortly (<5 s) before triggering the laser pulse. No more than four experimental trials were carried out with each preparation.
Low tension
rigor state was induced by relaxing the muscles in G10 and transferring
them to Ca-free rigor solution (G10 rigor) before
incubation in EGTA-buffered high Ca
(pCa 4.5
or 5.5) rigor solution. This solution contained 50 µM AP
A (Sigma), an inhibitor of myokinase activity. It
was finally replaced by photolysis solution containing 10 mM caged ATP, 10 mM EGTA, 6.1 mM
Mg
, 40 mM reduced
glutathione, 25 mM PIPES at pH 7.1, and proteinase inhibitors
as well as mitochondrial blockers (see above; Fig. 1B).
The free Ca
concentration in this solution was
adjusted to either pCa 4.5 or pCa 5.5.
When
thiophosphorylation of the 20-kDa myosin light chains was intended, the
muscles were incubated for an additional 10 min in 2 mM ATPS (Boehringer Mannheim) in high Ca
rigor
solution (pCa 4.5 or 5.5) before switching to photolysis
solution.
Figure 2:
pCa-tension curves obtained after
permeabilization with -escin or
-toxin, showing the effect of
[CaM] on steady state force. pCa-tension curves were
constructed from cumulative responses to stepwise increases in
[Ca
]
. The continuouslines show pCa-tension relationships in the
presence of (from right to left) 10 nM (
), 100 nM (
), 1 µM (
),
10 µM (
), and 40 µM (
)
exogenous CaM (n = 3). The dottedline reflects the Ca
sensitivity of strips
permeabilized with
-toxin (
), a preparation in which
endogenous CaM is retained (n = 4). Tensions are
normalized to the tension in pCa
4.5.
Increasing, at pCa 4.5, the exogenous CaM concentration from 10 nM to 40 µM did not change the steady state level of
tension, indicating that, although exogenous CaM increased the
sensitivity for Ca (Fig. 2), at this high free
[Ca
] it had no effect on maximum isometric
force.
We also determined the steady state Ca sensitivity of force in the presence of the endogenous CaM
concentration, by permeabilizing smooth muscles with Staphylococcus
aureus
-toxin. The pores produced by this toxin (1-2
nm) retain molecules (M
> 600-1000) like CaM
but are permeable to low molecular weight solutes (Cassidy et
al., 1979; Kitazawa et al., 1989, 1991). The pCa-tension curves of muscles permeabilized with
-toxin
lay between the pCa-tension curves obtained in the presence of
0.1 and 1 µM exogenous CaM in
-escin-permeabilized
strips (Fig. 2; pCa
= 6.2, n = 4), corresponding to [CaM]
0.25
µM. In contrast, the total [CaM] determined in
intact preparations was 37 ± 4 µM (n = 4).
To evaluate the
efficiency of photolysis, we calculated the concentration of
photoproducts according to Zucker and Steinhardt (1978) using the
dissociation constants determined by Ellis-Davies and Kaplan(1994) and
the initial and final [Ca]
measured by us. This calculation revealed that approximately 45%
of the total NP-EGTA was photolyzed.
As could be expected from the
steady state Ca-tension relationship, in the presence
of Ca
-loaded (pCa 7.0) NP-EGTA,
[CaM] >10 µM led to prephotolysis tension
that, in the presence of 100 µM CaM, reached up to
30-40% of F
(maximum force at pCa
4.5). After photorelease of Ca
, isometric tension
developed sigmoidally with an initial lag phase or delay (Fig. 3, Table 2). Comparison with the tension reached in
EGTA-buffered pCa 4.5 solution, recorded after the photolysis
experiments, showed that the plateau tension reached after photolysis
was at least 90% of maximum tension, except for the experiments
performed in the absence of exogenous CaM. In the latter case, force
usually reached variably lower plateau levels, consistent with the
steady state results at pCa 5.5 (Fig. 2). The time
required to attain 50% of the plateau tension (t
) was a function of the exogenous CaM
concentration (Fig. 4A), decreasing from 7.4 ±
0.6 s (0 added CaM, n = 5) to 4.3 ± 0.8 s (100
µM CaM, n = 5). This acceleration of force
development by CaM is sufficient to have been detected by (diffusion
rate-limited) activation of smooth muscle by increasing the
[Ca
] in solution (Kühn et al., 1990).
Figure 3:
The CaM
dependence of activation through photolysis of NP-EGTA. Preincubation
in the presence of exogenous CaM (40 µM) (after
permeabilization with -escin) reduces the delay and accelerates
contraction, as compared with the record obtained in the absence of
exogenous CaM (lowerforcetrace). Both curves are normalized to the respective peak tensions. Force
traces are superimposed on a recording of the
[Ca
]
transient produced by
photolysis of NP-EGTA, which was monitored using fluo-3 in the absence
of muscle, but under otherwise identical
conditions.
Figure 4:
A, CaM dependence of the reciprocal of tof contractions induced by photolysis of NP-EGTA
in muscles permeabilized with
-escin. B, dependence of
the delay (t
) on exogenous CaM following
permeabilization with
-escin and photolysis of NP-EGTA. Micromolar
concentrations of CaM reduce the t
of
contraction, but this effect of CaM saturates at approximately 40
µM with an EC
of 14
µM.
Exogenous CaM (2.5-100
µM) markedly shortened the delays of force development (t), measured as the time elapsing between t = 0 (photolysis) and the base-line intercept of a tangent
fitted to the steepest part of the tension trace, reducing t
from 470 ± 30 ms (2.5 µM CaM, n = 5) to 197 ± 22 ms (100 µM CaM, n = 5) (Fig. 4B). The effect
of CaM on t
saturated at about 40 µM.
The t
at 2.5 µM CaM was not
significantly different from the t
(470 ±
32 ms, n = 10) measured in the absence of exogenous
CaM. Photolysis of NP-EGTA in preparations permeabilized with S.
aureus
-toxin but under otherwise identical conditions
resulted in a delay of 565 ± 50 ms (n = 9), and t
of 11.7 ± 1.4 s (n = 7).
Figure 5:
Dependence of the kinetics of
phosphorylation induced by photolysis of NP-EGTA on exogenous CaM. The solidlines represent mathematical fits of the
experimental data to a single exponential function (n =
3-6). The apparent rate constants of MLC phosphorylation derived from these fittings are 1.1
s
(
, no added CaM), 0.9 s
(
, 5 µM exogenous CaM), and 2.9 s
(
, 40 µM exogenous
CaM).
Figure S13: Schemes I-III
To exclude the possibility that contaminant ATP caused MLC phosphorylation during the period of preincubation in the
photolysis solution, we also performed experiments adding 18 units/ml
apyrase to all rigor solutions and to the photolysis solution. This
treatment caused the force response to become transient upon
photolysis, as the result of ATP hydrolysis by apyrase, but had no
significant effect on the delay (n = 2, CaM
= 40 µM; data not shown). Furthermore, the
phosphorylation levels before, respectively, photolysis of caged ATP
and NP-EGTA, both in the absence of exogenous CaM, were not
significantly different (data not shown).
The time course of
MLC phosphorylation initiated by photolysis of caged ATP
was faster, even in the absence of exogenous CaM, than after activation
by photolysis of NP-EGTA in the presence of 40 µM CaM
(Fig. 6B, Table 3). The
fastest apparent rate constant of MLC
phosphorylation,
obtained by fitting the data to a single exponential function, was 3.5
s
. The rate of phosphorylation following photolysis
of caged ATP, much like the simultaneously monitored kinetic parameters
of force development, was independent of exogenous CaM ( Table 2and Table 3). However, the final phosphorylation
level (41%) was lower in the absence of exogenous CaM, corresponding
well to the lower levels of peak force. At [CaM]
= 5 µM, in spite of their different initial
rates (Table 3), the final levels of phosphorylation were not
significantly different following activation by photolysis of caged ATP
(56%) or NP-EGTA (57%).
Figure 6:
A,
comparison of the activation kinetics after liberation of,
respectively, Ca from NP-EGTA and ATP from caged ATP.
Contractions induced by activation with ATP from rigor are faster then
those induced by liberation of Ca
. Both the t
and the delay are shorter. Contractions were induced
successively in the same preparation at [CaM]
= 40 µM. Curves are normalized to peak
tension. Laser pulse at t = 0. B, time courses
of MLC
phosphorylation after activation by photolysis of
caged ATP in the presence of [CaM]
= 0
(
) or 5 µM (
). Note the faster time course
than observed after photolysis of NP-EGTA. Rate constants of
phosphorylation obtained from single exponential fits (solidlines) are 2.6 s
(no added CaM) and
3.5 s
(5 µM exogenous CaM) (n = 3-4).
Thiophosphorylation of MLC with
ATP
S, prior to photolysis of caged ATP, resulted in the fastest
rate of force development by shortening both t
and t
(Fig. 7; Table 2). t
decreased to 19 ± 2 (n = 6) and 32
± 5 ms (n = 6) at pCa 5.5 and 4.5,
respectively. We note that the shortest delay (19 ms) sets an upper
limit on the possible contribution of the series elastic element to the
lag phase (Horiuti et al., 1989) when contraction is initiated
from rigor. The time course of tension development was well fitted,
from t = 0.1 to t = 5 s, with two
exponentials with rate constants k
= 2.6
± 0.10 s
and k
=
0.4 ± 0.02 s
for experiments at pCa
4.5 and k
= 3.6 ± 0.29
s
and k
= 0.5 ±
0.02 s
at pCa 5.5, respectively. The
relative amplitudes of the faster components were 0.4 ± 0.03%
and 0.4 ± 0.02%. These t
and k
values at, respectively, pCa 4.5 and 5.5 are
significantly different (p < 0.05).
Figure 7:
Photolysis of caged ATP after
thiophosphorylation of MLC with ATP
S. Satisfactory
fitting of the initial 5 s of contraction requires the sum of two
exponential functions (dottedlines) with mean rate
constants of k
= 3.6 s
and k
= 0.5 s
at pCa 5.5. The fitted curve coincides with the experimentally
obtained curve. [CaM]
= 40
µM. The experimental trace is normalized to peak tension.
Laser pulse at t = 0.
Gel electrophoresis
of separate, identically treated preparations showed that
thiophosphorylation of MLC was 84 ± 6% (n = 3) before photolysis.
Figure 8:
A, comparison of the activation kinetics
following liberation of Ca in the presence of ATP
with activation through simultaneous release of Ca
and ATP by photolysis of NP-EGTA and caged ATP. The simultaneous
photolysis of NP-EGTA and caged ATP results in faster activation
kinetics than photolysis of NP-EGTA in the presence of MgATP. The two
records shown were obtained from the same preparation.
[CaM]
= 40 µM. Curves are
normalized to peak tension. Laser pulse at t = 0. B, the time course of MLC
phosphorylation after
activation from rigor by simultaneous photolysis of NP-EGTA and caged
ATP in the presence of 5 µM CaM
. Fitting
the data points to a single exponential function results in an apparent
rate constant of phosphorylation of 1.7 s
(n = 3-6).
We also measured the rates of
MLC phosphorylation to determine whether the faster
contractile kinetics following simultaneous photolysis of NP-EGTA and
caged ATP than of NP-EGTA alone were due to the different initial
states of cross-bridges (rigor and detached, respectively) and/or to
different rates of MLC
phosphorylation. In the presence of
5 µM CaM, MLC
phosphorylation increased from
1.6 to 36% with a pseudo-first-order rate constant of 1.7 ± 0.1
s
(Fig. 8B). This rate was
significantly faster than found after photolysis of NP-EGTA alone (0.9
± 0.1 s
), indicating that the acceleration of
force development was not solely due to the mechanical effects of
cooperative activation of nonphosphorylated by rigor cross-bridges
(Somlyo et al. (1988b); for striated muscle, see Weber and
Murray(1973), Goldman et al.(1984)).
The major findings of our study are the following: 1) the
delay (or lag phase) preceding force development is significantly
shorter when contractions are triggered by photolysis of caged ATP (in
the presence of Ca and CaM) than by photolytic
release of Ca
(in the presence of ATP and CaM), and
2) CaM can affect the initial rates of force development and MLC
phosphorylation without having a significant effect on the steady
state level of the latter. Because physiological contractions are
initiated by a rise in [Ca
]
, it
is likely that the mechanisms responsible for the delay (lag phase)
that follows photolysis of NP-EGTA also contribute to the physiological
activation delay (reviewed by Somlyo and Somlyo(1990, 1994)). We will
first consider the possible mechanisms contributing to the delay, based
on our experiments designed to reproduce the three activation sequences
shown in Fig. S13.
Fig. S13are components of the
physiological sequence (Fig. S13): a rise in
[Ca]
, the formation and binding
of Ca
CaM to MLCK, and production of the active
Ca
CaM-MLCK complex that phosphorylates myosin light chain
20 (MLC
) to permit activation of myosin ATPase by actin
(reviewed by Hartshorne(1987)). Fig. S13represents experiments
in which the muscles were stably thiophosphorylated (Cassidy et
al., 1979), and contraction was initiated from rigor by photolytic
release of ATP ( Fig. 7and Horiuti et al.(1989)),
bypassing MLC
phosphorylation and the steps preceding it.
Under these conditions, the release of ATP causes rapid detachment of
thiophosphorylated rigor cross-bridges followed by reattachment and
force development (Somlyo et al., 1988b; Horiuti et
al., 1989). Fig. S13represents the case of muscles
equilibrated with Ca
CaM to permit completion of
prephosphorylation reactions between Ca
, CaM, and
MLCK prior to photolysis; the release of ATP then causes
phosphorylation and contraction from an initial state of
dephosphorylated rigor bridges. Fig. S13I represents the delay
that follows the rise in [Ca
]
during physiological activation and includes Ca
binding to CaM, diffusion and binding of Ca
CaM to
MLCK, activation of the Ca
CaM-MLCK complex, and MLC
phosphorylation and contraction.
Of the steps represented in Fig. S13I the photolytic release of Ca (
68,000 s
) (
)and Ca
binding to CaM (t
2 ms; Kasturi et
al.(1993)) are very fast, as is the second-order rate constant
(10
M
s
) of
Ca
CaM binding to MLCK
(Török and Trentham, 1994).
According to this second-order rate constant, at pCa 5.5 in
the presence of 40 µM CaM (Ca
CaM
0.8
µM; average K
= 5
µM; Maune et al.(1992)), Ca
CaM will
bind to MLCK at a rate of approximately 80 s
, too
fast to make a significant contribution to the long (200 ms) lag phase.
Even slow diffusion hindered by binding (Rüegg et al., 1984; Tansey et al., 1994), is unlikely to
account for the entire delay, because increasing the CaM concentration
between 40 and 100 µM caused very little or no shortening
of t
(Fig. 4B).
Exogenous CaM (5 µM) shortened the
delay (t
) (Fig. 4B), but this
effect saturated (197 ms) at 40 µM compared with its value
(470 ms) at 2.5 µM CaM and in
-toxin-permeabilized
preparations (560 ms) containing endogenous [CaM]. This is
consistent with the conclusion that even under steady state conditions
only a fraction of total endogenous CaM (30-40 µM)
is available for contractile activation (Rüegg et al., 1984; Tansey et al., 1994; present study).
The lack of effect of less than 2.5 µM CaM on t
(Fig. 4B) contrasts with the
increased Ca
sensitivity and amplitude of steady
state force caused by 0.1 µM CaM (Fig. 2) and
suggests that steady state experiments overestimate the endogenous
[CaM] available for rapid activation by Ca
.
Indeed, at pCa 5.5 the effect of 1 µM CaM on
steady state tension was saturating or near saturating (Fig. 2),
whereas it was insufficient to affect t
. These
results and the long t
following photolysis of
NP-EGTA suggest that the [CaM] dependence of t
reflects the recruitment of CaM that is not readily available at
physiological [CaM] and/or some rate-limiting step(s) in the
activation of MLCK by Ca
CaM. The much lower CaM requirement
for maximal steady state force than for the rate of activation
indicates a significant contribution to t
by the
recruitment of slowly diffusible CaM. The Ca
sensitivity of steady state force (Fig. 2) and the long t
(565 ms) following photolysis of NP-EGTA in
-toxin-permeabilized smooth muscles suggest that the fraction of
total CaM that is rapidly available is even less than the available CaM
estimated from steady state experiments.
Diffusional recruitment is
unlikely to contribute to the lag phase at saturating [CaM].
Therefore, the relatively long t (
200 ms at
40-100 µM [CaM]), particularly when
contrasted with the short t
following photolysis
of caged ATP, suggests additional, nondiffusional contributions to t
. The possibility that following photolysis of
caged ATP the t
was short (48 ms at pCa
5.5) because the series elastic elements were already extended by the
initial, rigor tension can be excluded, because 1) there was a long
delay (197 ms) after photolysis of NP-EGTA even when prephotolysis
tension was high (30-40% of F
; see
``Results'') due to very high (100 µM)
[CaM], and 2) the initial rate of phosphorylation, which is
not affected by mechanical delays, was also significantly faster
following photolysis of caged ATP ( Table 3and Fig. 5and Fig. 6B). Furthermore, delays were significantly
different (48 versus 470 ms) when the prephotolysis levels of
phosphorylation were identical, in the absence of exogenous CaM ( Fig. 5and Fig. 6B). Therefore, we attribute the
short delay following photolysis of caged ATP at least in part to the
formation of active and available Ca
CaM-MLCK complexes
during incubation with Ca
and CaM prior to photolysis
(shown in Fig. S13), thereby shortening t
by an amount attributable to these prephosphorylation reactions.
The significant delay (about 200 ms) following photolysis of NP-EGTA at
saturating [CaM] suggests the contribution of additional
significant kinetic step(s), other than diffusion of CaM, to t
. Isomerization of smooth muscle
Ca
CaM-MLCK at approximately 1 s
(Török and Trentham,
1994) could account for, or at least contribute to, the nondiffusional
component of t
, but it is yet to be established
whether this isomerization is on the activation pathway.
Several of
our results (Table 2) could not be fitted to the model
(Hiromi(1979), cited in Horiuti et al., 1989), according to
which the lag phase reflects the kinetics of two sequential reactions,
MLC phosphorylation (``slow'') and transition
into force-generating states (``fast''), resulting in a delay
that approximates the t
of the faster of the two
processes, estimated from the rate of contraction of smooth muscle
containing thiophosphorylated MLC
(Horiuti et
al., 1989). However, fitting to this two-step model is appropriate
only when one of the two reactions is much faster than the other,
whereas in the present study the rates of MLC
phosphorylation (in the presence of 40 µM CaM) and
force development by thiophosphorylated cross-bridges were, in some
cases, similar ( Table 2and Table 3). It is apparent that,
depending on experimental conditions, the quantitative contributions of
different processes to t
are variable.
The rate, but not
necessarily the final level, of phosphorylation correlated well with
the rate of force development. For example, both rates were faster
following photolysis of caged ATP than of NP-EGTA in the presence of 5
µM CaM, although the plateau levels of MLC phosphorylation were not significantly different. Furthermore,
increasing [CaM] increased the rates of both contraction and
MLC
phosphorylation (Fig. 5) without necessarily
increasing their maximum amplitudes. These results do not support the
suggestion (Kühn et al., 1990) that the
acceleration of contraction by CaM in the absence of increased steady
state MLC
phosphorylation is mediated by a
phosphorylation-independent mechanism.
Following simultaneous
photolysis of caged ATP and NP-EGTA, the duration of the lag phase was
intermediate (t = 188 ms with 0 added CaM,
108 ms with 40 µM CaM) between the values obtained after
photolysis of either caged compound alone. The initial rate of
phosphorylation was also faster (1.7 s
versus 0.9 s
) following simultaneous (NP-EGTA/caged
ATP) photolysis than following photolysis of NP-EGTA alone at identical
(5 µM) [CaM]. These unexpected findings may have
been due to an inhibitory process during incubation with ATP, prior to
photolysis of NP-EGTA, that would be absent when ATP is released
simultaneously with Ca
. For example, ATP could
inhibit, directly or through phosphorylation of CaM, formation of a
putative preactive Ca
CaM-MLCK complex (Klee and Haiech,
1980). Phosphorylation of CaM reduces its activity in vitro (Sacks et al., 1992; Quadroni et al., 1994).
However, the use of a spinach CaM (40 µM) that lacks
several phosphorylation sites (Thr-26
Cys, Tyr-99
Phe,
Thr-146
Met; Watterson et al., 1980; Lukas et
al., 1984) did not accelerate contractions initiated by photolysis
of NP-EGTA (data not shown). Inhibition of CaM kinase II by KN-62 (20
µM) to prevent inhibitory phosphorylation of MLCK by CaM
kinase II (Tansey et al., 1994) also had no significant
effect. (
)
A second unexpected observation was that
increasing [Ca] from pCa 5.5 to pCa 4.5 prolonged both the lag phase and the t
of force developed following ATP release (Table 2). This slowing effect of high
[Ca
] was reversible and, therefore, not
attributable to Ca
-activated proteases. Inhibition of
MLCK could also not account for the slowing of the first phase of force
development (k
= 2.5 s
at pCa 4.5 versusk
= 3.6 s
at pCa 5.5) in smooth
muscles in which MLC
was thiophosphorylated.
[Ca
]
in smooth muscle normally
does not rise to pCa 4.5 (reviewed in Somlyo and Himpens,
1989), and the physiological significance and mechanism, whether
through binding to MLC
or action on thin
filament-associated protein(s), of the inhibitory effects of high
[Ca
] remain to be determined.
The major
contributor to the lag phase of activation of intact smooth muscle by
an agonist is the generation of inositol 1,4,5-trisphosphate preceding
the rise in [Ca]
(Somlyo et
al., 1988a; Miller-Hance et al., 1988; Somlyo et
al., 1992). We now conclude that the delay that follows the
increase in [Ca
]
(Somlyo and
Somlyo, 1994) is largely due to the combination of two mechanisms: 1)
recruitment of Ca
CaM from a slowly available CaM pool
(Rüegg et al., 1984; Tansey et
al., 1994; present study) and 2) activation of the
Ca
CaM-MLCK complex, possibly its isomerization
(Török and Trentham, 1994). We
also find that the rapidly available [CaM] is lower, that the
rate of phosphorylation of MLC
in situ can be
significantly faster than previous estimates, and that ATP may reduce
(slow) the availability of CaM for contractile activation.
Note Added in Proof-Western blots of
tissues obtained under our experimental conditions (45 min following
permeabilization with escin) showed no detectable loss of
endogenous calmodulin, compared with the content of nonpermeabilized
muscles.