RAPID COMMUNICATION
Force relaxes before the fall of cytosolic calcium in the
photomechanical response of rat sphincter pupillae
Andrew P.
Krivoshik1,2 and
Lloyd
Barr1
1 Department of Molecular and Integrative Physiology,
University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and
2 Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
In the rat
sphincter pupillae, as in other smooth muscles, the primary signal
transduction cascade for agonist activation is receptor
G protein
phospholipase C
inositol trisphosphate
intracellular
Ca2+ concentration ([Ca2+]i)
calmodulin
myosin light chain kinase
phosphorylated myosin
force development. Light stimulation of isolated sphincters pupillae
can be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMR
ideal for testing models of regulation of smooth muscle myosin
phosphorylation. We measured force and
[Ca2+]i concurrently in sphincter pupillae
following stimulation by light flashes of varying duration and
intensity. We sampled at unusually short (0.01-0.02 s) intervals
to adequately test a PMR model based on the myosin phosphorylation
cascade. We found, surprisingly, contrary to the behavior of intestinal
muscle and predictions of the phosphorylation model, that during PMRs
force begins to decay while [Ca2+]i is still
rising. We conclude that control of contraction in the sphincter
pupillae probably involves an inhibitory process as well as activation
by [Ca2+]i.
contractile force; light; contraction; myosin phosphorylation; contraction models
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INTRODUCTION |
ABSORPTION OF LIGHT
BY photosensitive receptor molecules in sphincter pupillae smooth
muscle myocytes evokes contraction by triggering the release of calcium
ions (Ca2+) from intracellular stores. This photosensitive
receptor, which has an action spectrum similar to the absorption
spectra of rhodopsin, is located in the plasma membrane and has
antigenic sites, which bind antibodies for rhodopsin (1).
The photomechanical response (PMR) signal transduction cascade couples
an opsin receptor via a G protein to the typical smooth muscle
phosphorylation contraction cascade. The member of the opsin family in
the cell membrane of the sphincter pupillae myocytes that triggers the
PMR is probably either rhodopsin (2) or melanopsin
(22). It has been demonstrated that the PMR of the
isolated pupillary sphincter is independent of the retinal pretectal
reflex pathways, which mediate the pupillary reflex of humans and other
mammals. In frog sphincter pupillae, the PMR and the neural pupillary
reflex are equally strong (1). However, in mammals, the
PMR is much weaker than the contraction elicited by muscarinic receptor
activation. Nonetheless, forces developed during mammalian PMRs,
1-50 µN, are easily measured. The PMR of the frog sphincter
pupillae is a fast relatively simple smooth muscle contraction. It
shows none of the "latch" properties commonly found in other smooth
muscles. Most significantly, the force time courses of frog PMRs to
light stimuli of different durations or intensities are quantitatively
predicted by a simple model of the phosphorylation cascade
(3). We report here for the first time, additionally, the
time courses of cytosolic Ca2+ concentration
([Ca2+]i) during PMRs. We studied the PMR of
sphincter pupillae from albino (4, 17) rats
to minimize any interference with our fluorescence measurements by
melanin, present in the myocytes of the frog sphincter pupillae. We
found the first direct evidence of a very fast regulatory component in
addition to the Ca2+-calmodulin-phosphorylation cascade in
smooth muscle.
We conclude that, while for some other mammalian smooth muscles
alternative activating pathways have been proposed, a fast Ca2+-independent inhibition is most likely responsible for
the termination of contraction in the sphincter pupillae.
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METHODS |
Albino Sprague-Dawley rats were maintained in the dark for at
least 10 days to increase their sensitivity to light. Sphincter pupillae preparations were isolated under a dissecting microscope in
far red light (Wratten Filters 1A) at 0°C. The annular bundles of
smooth muscle were loaded with indo 1 by exposing them for 40 min to 2 µM indo 1-acetoxymethyl (AM) (9, 29,
30). To augment loading, 10 µl of 20% (wt/vol) Pluronic
F-127 dissolved in dimethyl sulfoxide was added to the 2.5-ml mammalian
isotonic phosphate-buffered physiological bathing solution [136 mM
NaCl, 1 mM MgSO4· 7H2O, 0.1 mM EDTA, 4 mM
propionic acid (hemicalcium salt), 5 mM glucose, 2 mM sodium
succinate·7H2O, 1.6 mM
NaH2PO4·H2O, 6.8 mM
K2HPO4·3H2O] in the tissue
chamber. EDTA is used as a chelating agent to remove contaminant heavy
metals from solution. To minimize extracellular hydrolysis of AM ester,
a phosphate buffer is used instead of an amine buffer. The isolated
sphincter's pupillae in a 37°C chamber on the stage of a Nikon
Optiphot microscope were attached to a Cambridge 400A tension
transducer lever. The microscope system utilizes two Hamamatsu R374
head-on type photomultiplier tubes and custom band-pass filters. The
relative dead time of the electronics system (including photomultiplier
tubes) dedicated to the fluorescence measurements was experimentally
determined to be on the order of milliseconds and estimated to be no
greater than 10 ms. The microscope setup was extensively modified with light traps to minimize stray light, since the background contractile activity of the sphincter pupillae is proportional to the logarithm of
background light intensity (2). Filters were used to allow excitation of the indo 1 without stimulating the sphincter pupillae and
also to stimulate the sphincter pupillae to produce a PMR without
interfering with the fluorescence measurements. Light that stimulated
the preparation passed through a long-pass dark orange filter (OG515)
and was strongly attenuated by the filters in the
photomultipler light paths. To excite the indo 1 without appreciably
eliciting a PMR, shorter wavelength of light outside of the action
spectra of the PMR was used to excite indo 1. This was done by strongly
attenuating visible light in the exciting light path with a 310-nm
filter with half-band-pass width of 50 nm in series with a 347-nm
band-pass filter 40 nm wide at half band pass. Both the force signal
and fluorescence signals are digitally sampled and processed through
digital Savitsky-Golay smoothing filters (12,
24). The fluorescence signals are subsequently manipulated
through a ratiometric data analysis to generate an apparent
Ca2+ measurement (9). Digital data sampling
began 0.5 s before the beginning of an experimental flash stimulus
to sample the dark steady state before flash stimulation.
Because the structural apparatus for force transmission in smooth
muscle occupies between 80 and 90% of the cell volume
(8), contraction reflects the spatial average of
[Ca2+]i (20). In fact, the
spatial average of [Ca2+]i and
Ca2+ subjacent to the cell membrane may even be
anticorrelated (32). Therefore, fast time resolution of a
spatial averaged [Ca2+]i and force were used
to test quantitative signal transduction model predictions
(15, 16).
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RESULTS |
The effects on the PMR of varying the intensity of light
with the same durations of illumination were tested. Figure
1 shows that the PMR has several unusual
kinetic properties. First, Fig. 1, A and B, shows
the similarities of concurrent time courses of developed force and
[Ca2+]i in a sphincter stimulated by two
stimuli of 0.5 s duration, but different intensities. Intensity
increases the amplitude of the response but does not change the time
course. Second, the latency period of the rat PMR is much shorter than
that of the amphibian PMR (2) or fish PMR
(1). After brief flashes of light, the peak force of a PMR
occurs after the stimulus light flash has ended, i.e., the flash of
light triggers a cascade perturbation that continues to activate
Ca2+ and force after the light stimulus is terminated.
Finally, maximal peak force occurs before maximal peak
[Ca2+]i, and the force decays faster than
does the [Ca2+]i.

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Fig. 1.
Force and intracellular Ca2+ time courses
during 2 photomechanical responses (PMRs) of an albino rat
sphincter pupillae stimulated by 2 light flashes of same duration but
different intensities. A: 2 force time courses following
0.5-s flashes whose intensities were attenuated by neutral density
filters: 0.2 optical density (OD) or 0 OD (no filter), respectively.
Very large forces (>10 mg), occurring during peak of largest PMR were
out of dynamic range of analog-to-digital converter and were digitized
manually from parallel records obtained via an analog strip chart
recorder. Peaks of responses occur with same latency after each
stimulus. B: 2 intracellular Ca2+ time courses
corresponding to force time courses in A. Peaks of
intracellular Ca2+ time courses all occur at same interval
after stimulus, but a little later than the time to peak for the force
trajectory. Similarly, the decay of intracellular Ca2+ from
its peak is slower than the decay of force from its peak. Amplitudes of
force and Ca2+ time courses increase less than linearly
with increasing stimulus intensity. From studies with slower sampling
rates over longer periods of data acquisition, Ca2+ for
comparable stimuli returns entirely to baseline on the order of 2 min.
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If the stimulus flash duration and quantal contents are small enough,
there is reciprocity between intensity and duration, and the responses
to such constant quanta, variable duration flashes, are
indistinguishable. As seen in the amphibian PMR, the force of
any particular sphincter's response to a short flash of light is
proportional to the logarithm of intensity times duration, namely the
quantal content (3).
Therefore, PMRs to 0.5-s flashes of different intensities differ only
in amplitude, with the shape of the time courses of the PMRs appearing
independent of the light intensity. Therefore, the force time courses
normalized to the peak force are nearly identical and superimpose (Fig.
2A). Thus
the time it takes to reach any given fraction of the peak force of the
twitch is independent of stimulus intensity. Therefore, also, the
exponential time constants for the evolution and decay of the PMR vary
little with intensity. In Fig. 2A, the corresponding
[Ca2+]i time courses normalized to peak
[Ca2+]i may be seen to also collapse to a
single curve. The exponential time constants for the evolution and
decay of the [Ca2+]i time course therefore
also vary little with intensity. The semilogarithmic scale for the
normalized force and [Ca2+]i time courses
makes the time constants of the signal transduction processes more
apparent. The rise of force is nearly a single exponential, but the
relaxation involves at least two exponential components. As is also
shown in Fig. 2A, there are both very fast and slow
components to the relaxation of [Ca2+]i as
well. The complex relationship in the dynamic state between [Ca2+]i and force are apparent in the phase
relationship of [Ca2+]i with respect to force
as shown in Fig. 2B. During the rapid falling phase of force
in a PMR, there is a clear decoupling from still slowly rising
[Ca2+]i. The family of phase loops generated
by increasing flash intensity with constant duration are clockwise in
time and also when normalized to peak collapse to a single canonical
phase loop.

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Fig. 2.
Relaxation of force precedes fall of intracellular
Ca2+ PMRs of sphincter pupillae smooth muscle of albino
rat. Moreover, trajectories of PMRs to short flashes of increasing
intensity elicit responses whose amplitudes increase but whose time
courses remain the same. Peaks of force and intracellular
Ca2+ occur at times that are characteristic of stimulus
duration, not stimulus strength. Phase relations between force and
intracellular Ca2+ are therefore the same for all PMRs
resulting from same duration stimuli. A: overlay of semilog
plots of normalized force and normalized Ca2+ during PMRs
after 0.5-s flashes of different intensities (data from Fig. 1).
Normalized force time courses superimpose as do normalized
Ca2+ time courses. Both force and Ca2+ time
courses have 2 time constants apparent in relaxation phases. The 2 dominant relaxation time constants for force are in the neighborhood of
2.8 and 9.8 s. The 2 dominant relaxation time constants for
Ca2+ are in the neighborhood of 7.7 and 14.7 s.
B: phase relationship between force and intracellular
Ca2+ shows time increases clockwise around the hysteresis
loop (data from Fig. 1). Because relaxation is slower than contraction,
there are many more data points in the relaxation portion of the
hysteresis loop. Clearly, force is falling in a period, upper right
hand quadrant, where intracellular Ca2+ is still rising.
Thus, in time, intracellular Ca2+ changes first lead, and
then lag behind force changes during a photomechanical response. Return
to baseline force and baseline Ca2+ is represented as
completion of hysteresis loop in phase diagram. C: longer
durations of stimulating flashes elicit larger PMR responses up to at
least 10 s, but responses evolve toward a plateau. Three time
courses of force during PMRs in response to light flashes of 2, 5, and
10 s. Quanta of light arriving later during a flash longer than
~0.3 s have less effect on increasing force and intracellular
Ca2+. During very long flashes force slowly declines due to
use decline (see Fig. 5). D: similarly, 3 time courses of
intracellular Ca2+ during PMRs (shown in C) in
response to light flashes of 2, 5, and 10 s. In each case, decay
of intracellular Ca2+ is slower than decay of force.
Initial spurious points at beginning of PMRs attributable to
stimulating light flash (shutter) artifact were deleted. E:
phase relationship between force and intracellular Ca2+
shows time increases clockwise around the hysteresis loop (data from
C and D).
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Figure 2, C and D, shows the time courses of PMRs
to stimuli of constant intensity and varying duration. Responses to
long flashes tend toward a plateau after ~15 s. The family of phase loops generated by increasing flash duration with constant intensity are clockwise in time and follow similar initial rise trajectories and
final relaxation trajectories, except that the maximal Ca2+
maximal force shoulder is displaced to higher values for longer durations. Moreover, the phase relationships follow similar initial and
final trajectories and are all clockwise in time (Fig. 2E). Qualitatively similar clockwise phase loops are found in stimulating flashes as long as 1 min.
To check whether this response of the rat sphincter pupillae is
peculiar, we did similar experiments on the frog sphincter pupillae.
Figure 3 is a semilogarithmic plot of
force and [Ca2+]i time courses for the
average of five frog PMRs to flashes of 0.1 s duration. In
contrast to the rat PMR, both the rise and relaxation of force in the
frog appear to be simple exponential functions of time
(2). Nonetheless, as shown in Fig. 3B, in the
frog PMR, the phase relationship of [Ca2+]i
with respect to force follows a trajectory similar to that of the rat
but with slower kinetics. In the phase diagrams of Fig. 2, B
and E, and Fig. 3B, time increases clockwise
around the hysteresis loop, emphasizing that force falls before
[Ca2+]i. By using very short (0.1-s) flashes
of light, the shutter artifact could be separated in time from the
recordings of [Ca2+]i and force. The initial
rise of [Ca2+]i is seen to precede the rise
of force (Fig. 3B).

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Fig. 3.
Relaxation of force precedes fall of intracellular
Ca2+ PMRs of sphincter pupillae smooth muscle of frog.
Averaged normalized intracellular Ca2+ and normalized force
time courses from 5 frog PMRs after 0.1-s flashes of constant intensity
are slightly slower than rat. Note however, frog sphincter pupillae
were investigated at 25°C. Due to intrinsic pigments in the frog
preparation, in addition to signal averaging, it was necessary to
increase loading time, concentration of indo 1-AM, and intensity of
short-wavelength light used to excite indo 1. A: PMRs of
frog sphincter pupillae are qualitatively similar to those of rat. In
frog, as well as rat, force begins to fall while Ca2+ is
still increasing. In frog PMR, there is only 1 apparent time constant
in relaxation of both force and Ca2+ and is, therefore,
kinetically less complex than rat PMR. B: for frog PMRs,
phase relationship between force and intracellular Ca2+
also shows time increases clockwise around the hysteresis loop (data
from A).
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To test whether or not force relaxation generally precedes fall of
[Ca2+]i in other smooth muscles, we measured
[Ca2+]i and force concurrently in
spontaneously active rat duodenal muscle rings. These data show that
force lags [Ca2+]i by a nearly constant phase
angle. In particular and in contrast to the PMRs, a fall in
[Ca2+]i always precedes the fall in force
(Fig. 4A). Figure
4B shows the phase relationship in which time increases
counterclockwise around the hysteresis loop and the phase plot is close
to a circle. Ca2+ and force data from similar experiments
described in the literature (21) transform into
qualitatively similar phase diagrams. Similar counterclockwise phase
diagrams have been reported for vascular smooth muscle
(10). The differences between sphincter pupillae and
intestine predict a qualitatively different signal transduction system
in the sphincter pupillae, which allows the pupil to relax rapidly to
decreases in ambient light.

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Fig. 4.
Intracellular Ca2+ changes precede force
changes throughout contractile cycles of rhythmic contractions of rat
duodenum. Strips of rat duodenal circular muscle with longitudinal
layer attached were mounted and loaded in same manner as sphincters
pupillae. A: during spontaneous rhythmic contractions, force
clearly lags behind intracellular Ca2+ changes. In absence
of appropriate calibrations, ratio of emitted fluorescent light at
405-485 nm corrected for background fluorescence is reported
instead of actual Ca2+ concentration. B: in
contrast to results from sphincters pupillae, intestinal muscle relaxes
only after intracellular Ca2+ falls. For these rhythm
contractions, phase relationship between force and fluorescence ratio
shows time increases counterclockwise around the hysteresis loop (data
from Fig. 3A). , Fluorescence ratio and force of native
data averaged over the 6 cycles. Because actual Ca2+
concentration is a monotonic function of fluorescence ratio, the shape
of the hysteresis loop would be changed by calibration but not the
direction that time takes around the loop. From these observations for
duodenal muscle, there is no need to posit any modulator of force
beyond intracellular Ca2+ concentration.
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Given the rapid response time of the experimental apparatus and the
rapid reaction kinetics of indo 1, it is unlikely that such a phase
relationship between Ca2+ and force is a measurement
artifact attributable to the system response time or kinetic properties
of the fluorescent Ca2+-sensitive probe. Indo 1 is well
suited to measure rapid changes in concentration of intracellular
Ca2+; stopped flow data for indo 1 indicate that the
association rate constant approach the diffusion-controlled limit, and
the measurement of a transient decay in Ca2+ with a
half-time on the order of 15 ms is possible in a system with a dead
time on the order of 1 ms (11). Furthermore, when ultrashort duration flashes of stimulating light are used (Fig. 3B), the latency in the initial rise of force after the
initial rise in Ca2+ is consistent with the rapid response
time of the experimental apparatus. Although both rate constants and
dissociation constant (Kd) are
essentially invariant between pH 7.0 and 8.0 (11), undetected transient changes in pH below that range may distort the
apparent Ca2+ kinetics (19). Although indo 1 (9, 29) is rather selective for
Ca2+, other potential factors such as Mg2+ and
Zn2+ transients and fluorescent probe protein interactions
may also confound the interpretation of the Ca2+ kinetics.
As an experimental instrument control, it is notable that measurements
in the intestinal preparation (Fig. 4) compare well with those in the
literature (10, 21).
During control experiments in the rat sphincter pupillae, it was noted
that PMRs decayed in amplitude as the experiments proceeded. The
maximal peak amplitude in the PMRs of a preparation follows a nearly
perfect exponential decay as a function of number of light stimuli of
fixed duration and intensity. The decay of peak response is essentially
independent of between flash recovery time and hence also independent
of total time of survival (Fig. 5A). The decay is a function
of both the intensity and duration of stimuli. The greater the duration
of the flash stimulus, the greater the rate of decay (Fig.
5B). After many flash stimuli, although the maximal peak PMR
is attenuated, the response to an acetylcholine challenge remains
essentially unchanged, so that the decline with frequency of
stimulation is related to the early light receptor-like portion of the
signal transduction pathway. Therefore, small duration and low
intensity flashes were typically used during most Ca2+
measuring experiments. Long exposure to mercury light used to excite
indo 1 causes tissue damage, and therefore Ca2+ could not
be meaningfully measured concurrently with force for these decay
experiments. Also, since PMRs normalized to peak tension have the same
time courses, myosin phosphatase is probably not involved. Attempts to
treat the preparation with all-trans-retinol were not
protective. Preliminary experiments show that both caffeine and okadaic
acid increased this decay, suggesting that phosphorylation of the light
transduction system may be responsible for this decay, in that
phosphorylation may inactivate rhodopsin (5). Moreover, perhaps this decay phenomenon is related to a regulatory protein such
as arrestin (18), since transgenic mouse rods that lack arrestin exhibit prolonged photoresponses (31). So,
although interesting, this usage decay phenomenon is probably
independent of the second regulatory process of the PMR.

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Fig. 5.
Responsiveness of rat sphincter pupillae decays in vitro
according to usage. A: force amplitudes of PMRs decay
exponentially with number of flash stimuli. Two sphincter pupillae from
the same rat were stimulated with 0.5-s duration, constant intensity
flashes. For 1 sphincter the recovery time between flashes was first 3 min for 10 flashes and then 5 min for three flashes. For the other
sphincter the recovery time between flashes was first 5 min for 9 flashes and then 3 min for 10 flashes. On a semilog plot all the force
amplitudes fall on parallel straight lines; the slopes of the 4 best
fit lines for each subgroup are not distinguishable by Chi square
(P > 0.95). Varying recovery time between flashes does
not alter decrement in responsiveness following a PMR; instead it is
proportional to the PMR amplitude. B: decrement in
responsiveness after a PMR increases with duration of the stimulus and
response. A sphincter pupillae was stimulated with flashes of the same
intensity but whose durations first 0.1 s and then 0.2 s with
a 3-min interflash recovery delay. Doubling flash duration increases
the decrement per PMR in addition to increasing initial peak force
generated. These 2 slopes are statistically different to 99%
confidence by homoscedastic t-test for mean differences.
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DISCUSSION |
The relation between force and Ca2+ in smooth muscle
varies during a contraction-relaxation cycle, and the apparent
Ca2+ sensitivity of force depends on the mode of activation
(26,27). Clearly, the relative activities of
myosin light chain kinase and the myosin phosphatases determine the
level of myosin phosphorylation. Therefore, a decrease in force at
unaltered Ca2+ may be attributable to factors that alter
the activities of myosin light chain kinase or the phosphatases, such
as calmodulin (CaM) kinase II, telokin, and cGMP-activated kinases.
Relaxation disproportionate to the decline in
[Ca2+]i has been previously related to rapid
dephosphorylation of regulatory myosin light chains, due to either
inactivation of myosin light chain kinase by CaM kinase II
(28) or enhanced phosphatase activity referred to as
"desensitization to [Ca2+]i"
(25). Decreased myosin phosphorylation accompanying a
decline in force during maintained high
[Ca2+]i has been observed in permeabilized
smooth muscle (13). Moreover, CaM kinase II has been shown
in vitro to decode the frequency of Ca2+ spikes into
distinct kinase activity periods (6). Thin filament proteins, such as calponin and caldesmon, may also influence the relation between force and Ca2+. Mitogen-activated protein
kinase in vascular smooth muscle may be related to
Ca2+-independent smooth muscle contraction and caldesmon
phosphorylation (7). In uterine smooth muscle, oxytocin
receptor stimulation coupled via a G protein activates phospholipase C,
generating inositol trisphosphate that triggers release of
Ca2+ from intracellular stores; this oxytocin receptor
stimulation of phospholipase C is inhibited by cAMP involving the
action of protein kinase A (23). In general, if force is
proportional to myosin phosphorylation, then the clockwise in time
phase diagram between Ca2+ and force implies the existence
of an inhibitory pathway in the contraction cycle. Concurrent
measurements of myosin light chain phosphorylation might discriminate
between such putative mechanisms, but current methods are not adequate
to resolve such fast time courses.
Preliminary experiments with rat PMR show that cAMP but not cGMP
attenuates the PMR as well as increases the rate of PMR relaxation (14). However, cAMP collapses the phase loops to the right
(14, 15). If there is only a
Ca2+-dependent activating process, then the direction of
time in the phase diagram would be counterclockwise. If there is a
strong enough Ca2+-independent inhibition during a
relaxation, then, however, the phase diagram may run clockwise. A
constant increase in the relationship of Ca2+ to force may
shift the phase diagram to the right. If cAMP is present in an
inhibitory pathway, then added cAMP should collapse phase loops
progressively (i.e., if the cAMP process is saturated, cAMP release
during a cycle would have no effect). Although cAMP may activate myosin
phosphatases, cAMP may also increase phosphorylation of myosin light
chain kinase. Although the appearance of an inhibitory process (e.g.,
via a cAMP event) to initiate the fall of force seems possible
(15), other possibilities exist. If light in parallel to
triggering Ca2+ release also turned on a faster pathway
that increased force development or the efficacy of Ca2+
(e.g., via a GTP mechanism), then, when light turns off, that activating system's quick collapse might cause force to fall before the slower Ca2+ cascade collapses. A third possibility also
might involve cAMP but in a manner similar to cGMP's role in the
retinal rod. If there is a significant concentration of cAMP causing a
tonic inhibition of the contractile cascade and if also light quickly
turned on a disinhibiting phosphodiesterase, then, when the light is
turned off, a cAMP-induced inhibition might reappear before the slower Ca2+ cascade collapses and
[Ca2+]i begins to fall. Some of these
alternatives can be tested by investigating the pharmacological aspects
of GTP and cAMP behavior in rat sphincter pupillae smooth muscle.
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ACKNOWLEDGEMENTS |
This work was supported in part by National Science Foundation
Grant DCB-41526, by the University of Illinois at Urbana-Champaign Research Board, and by National Institute of Drug Abuse National Research Service Award Grants F30-DA-05574 and T32-GM-08276.
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FOOTNOTES |
Address for reprint requests and other correspondence:
A. P. Krivoshik, Pediatrics-Baldwin 3B, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: krivoshik.andrew{at}mayo.edu).
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.
Received 14 February 2000; accepted in final form 4 April 2000.
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