From the Wolfson Institute for Biomedical Research, University College London, Cruciform Building, Gower Street, London WC1E 6BT, United Kingdom
Received for publication, July 26, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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Soluble guanylyl cyclase (sGC) catalyzes cGMP
synthesis and serves as a physiological receptor for nitric oxide (NO).
Recent evidence indicates that key properties of sGC within cells
differ from those of purified sGC. We have devised a technique for
resolving NO-stimulated sGC activity in cells on a sub-second time
scale, enabling the first quantitative description of the kinetics of the enzyme within its natural environment. Upon release of NO from a
caged derivative, sGC became activated without any lag observable at a
20-ms sampling time. Deactivation of sGC on removal of NO occurred with
a rate constant of 3.7 s Nitric oxide (NO)1
performs diverse biological functions, ranging from relaxation of
smooth muscle and inhibition of platelet aggregation to neural
signaling in the peripheral and central nervous systems (1, 2). The
principal receptor mediating the physiological actions of NO is the
cGMP-synthesizing enzyme, soluble guanylyl cyclase (sGC). Exposure to
NO increases the sGC catalytic activity up to several hundred-fold
(3-5), and the resulting cellular accumulation of cGMP can engage a
number of downstream targets, including cGMP-dependent
protein kinase (6), cGMP-regulated phosphodiesterases (7), and cyclic
nucleotide-gated ion channels (8) to bring about the various biological effects.
Since the purification of sGC in the late 1970s, much knowledge of the
structure and the mechanism of activation of the enzyme by NO has
accrued (9). The enzyme is an As with any other signaling molecule, knowledge of the kinetic
properties of the receptor is a prerequisite for understanding how the
signals are decoded. When studied in tissue homogenates or in its
purified form, sGC exhibits simple Michaelis-Menten-type kinetics.
Consequently, in the presence of NO, excess substrate (GTP), and
cofactor (Mg2+), sGC synthesizes cGMP at a constant rate
over long periods, more in the manner of a "housekeeping" enzyme
than a receptor. However, our recent investigation (11) of how sGC
behaves within its natural cellular environment suggested a very
different profile of activity; within seconds of adding NO, the enzyme
underwent rapid desensitization to reach a steady-state level of
activity that was 10-15% of the peak. Other kinetic properties of sGC
were also different in the cells compared with those of the purified enzyme, notably the potency of NO and the rate at which the enzyme deactivated following removal of NO.
The precision of this previous study was handicapped by the poor
temporal resolution of the method used (simple manual pipetting), and
so much of the information had to be extracted from the data by
mathematical deconvolution techniques. Furthermore, the values of key
kinetic parameters were not disclosed because they were too fast to be
measured. To address these deficiencies, we have devised a method for
investigating second messenger responses in cells on a sub-second time
scale. The aim was to determine the kinetic constants for sGC
activation, deactivation, and desensitization, and so obtain the first
quantitative description of NO-stimulated sGC activity in a
physiologically relevant setting.
Cerebellar cell suspensions were prepared from 8- to 9-day-old
rats as described previously (11) and were incubated at a concentration
of 20 × 106 cells/ml at 37 °C. From at least
1 h before use, the NO synthase inhibitor
L-nitroarginine (100 µM) was included to
prevent possible complications arising from endogenous NO production.
Aliquots (200 µl) of the cells were added to a glass vessel (V005;
QMx Laboratories, Essex, UK) and preincubated for 5-10 min
with the phosphodiesterase inhibitor sildenafil citrate (100 µM). This concentration is sufficient to inhibit
completely phosphodiesterase activity in these cells at the cGMP levels
achieved in the present experiments (12). The vessel was placed in the
rapid-quenching apparatus (Fig. 1) after addition of the UV-sensitive
compound potassium ruthenium nitrosyl pentachloride (13), hereafter
referred to as "caged-NO," to the suspension.
Rapid Quenching of Cells--
The apparatus (Fig. 1a)
consists of a perspex module, with a central port surrounded by a water
jacket at 37 °C. The glass vessel sits within the port, over a
photodiode (OSD15-E visible light enhanced; RS Components, Northants,
UK). Above the vessel, a XF-10 xenon flash lamp (Hi-Tech Scientific;
Salisbury, UK) fitted with a UG11 filter (peak transmission 320 nm) was
mounted at a distance of ~15 cm from the bottom of the vessel. The
focal point for the lamp was only 2.5 cm so that, on flashing, the cell
suspension was exposed to an unfocused pulse of UV light. Just above
the vessel, either one or two glass pipettes (tip internal
diameter ~ 350-400 µm), formed with an electrode puller, were
positioned obliquely with micromanipulators. These pipettes were
attached to a Picospritzer (Picospritzer IID, General Valve Corp.).
The principle of the machine is simple; at a predetermined time after
uncaging NO with an UV flash (intensity, 200 V, duration 1 ms), a
volume (20 µl) of 3 M trichloroacetic acid is expelled from the pipette under pressure (50 pounds/square inch, 10-ms pulse
duration) and mixes with the cell suspension (final trichloroacetic acid concentration = 0.3 M), denaturing the cells and
therefore quenching the cGMP accumulation. The photolysis time of the
caged-NO is <1 ms (13) and estimated quenching time is <3 ms (14). The triggering of the flash lamp and Picospritzer is coordinated by a
Master 8 voltage step generator (Intracel Ltd., Hertfordshire, UK). The
vessel was then removed from the apparatus, and the sample was
neutralized with an equal volume of
1,1,2-trichlorofluoroethane:tri-n-octylamine (4:1 v/v),
mixed thoroughly and spun at 2500 × g for 10 min. The upper (aqueous) layer was removed and its cGMP content measured by radioimmunoassay.
To evaluate the method in terms of speed and thoroughness of mixing, 20 µl of India ink was injected into 200 µl of incubation buffer. The
resulting change in light intensity was registered as a change in
potential difference (P.D.) across the photodiode (response time 12 ns). The P.D. was monitored with an oscilloscope and captured on
computer. The mean change in P.D. with time is illustrated in Fig.
1b. There was a 15-ms delay from triggering of the
Picospritzer to onset of mixing (presumably attributable to the time
taken for the propagation of the pressure pulse). The subsequent total
mixing time was ~5 ms. In the data presented, all time points take
the 15-ms lag into consideration (i.e. they represent the
time of onset of mixing).
In experiments aimed at determining the rate of sGC deactivation, a
second pipette was used to inject either incubation buffer (for control
cells) or hemoglobin (final concentration 100 µM) to
scavenge NO. With this excess of Hb over NO, free NO would disappear
with a half-time of less than 50 µs (15). The cells were subsequently
quenched with trichloroacetic acid at fixed intervals.
The data are presented as means ± S.E. for 3-4 independent
samples at each time point in each experiment.
For identifying cGMP accumulating cells by immunocytochemistry,
paraformaldehyde was injected in place of trichloroacetic acid, to give
a final concentration of 4%. The samples were allowed to fix for 15 min, and they were then processed as described previously (11), except
that the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) was present
in the mounting medium to illustrate the total cell number in a given
field. Slides were viewed with a confocal microscope (TCS SP; Leica,
Heidelberg, Germany).
Estimation of NO Released from Caged-NO--
To estimate the
concentration of NO achieved following flash photolysis, the method of
Murphy et al. (13) was adapted. Caged-ATP (ATP,
P31,-(2-nitrophenyl)ethyl ester, disodium salt) was added
to 200 µl of freeze-fractured cells, which had been boiled for 20 min, sonicated, and vortex-mixed. Following flash photolysis, the
samples were kept on ice, and the ATP was assayed (in subdued light) by the firefly luciferase method ("ATP-lite-M" kit, Packard Instrument Co.). Un-flashed samples were assayed as a control, and the values were
deducted from the total ATP in the samples, to give the concentration of uncaged ATP. As the caged-ATP and caged-NO compounds have nearly identical absorption spectra in the range of filtered flash lamp wavelengths, the amount of uncaged NO can be deduced from the relative
quantum efficiencies of the cages, which is 0.08 (13). With the above
protocol, 2.96% of ATP was uncaged per flash, indicating that 0.237%
of NO would be uncaged. This value was used for estimating the NO
concentrations achieved in the experiments.
Materials--
Potassium ruthenium nitrosyl pentachloride and
ATP, P31,-(2-nitrophenyl)ethyl ester (disodium salt) were
from Molecular Probes (Leiden, The Netherlands);
L-nitroarginine was from Tocris Cookson (Bristol, UK);
sildenafil citrate was supplied by the Chemistry Division, Wolfson
Institute for Biomedical Research. All other special chemicals were
from Sigma.
The purpose-built rapid quenching apparatus (Fig.
1 and "Experimental Procedures")
allowed the termination of cGMP accumulations in the cell suspensions
with a mixing time of ~5 ms and quenching time <3 ms. In applying
the technique to the measurement of sGC kinetics, therefore, an
operational limit of 20-ms intervals between data points was
imposed.
1, which is
25-fold faster than the fastest estimate for purified sGC.
Desensitization of sGC occurred with a time constant of 6.9 s at
an estimated 70 nM NO and became faster at a higher
concentration, indicating that NO accelerates desensitization. The
concentration-response curve for NO consequently became increasingly
bell-shaped with time, a phenomenon that causes the apparent potency of
NO to increase with time. The results indicate that sGC within cells
behaves in a highly dynamic fashion, allowing the NO-cGMP pathway to
operate within a kinetic framework more resembling that of
neurotransmission than the properties of purified sGC suggest.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-heterodimer with a prosthetic heme
group, the NO binding site, attached to the
-subunit. Somewhat
unexpectedly by comparison with analogous receptors, sGC exhibits
limited molecular heterogeneity; only 2
- and 2
-subunits have
been identified, and only 2 isoforms have so far been found to exist at
the protein level as follows:
1
1, which
is widespread, and
2
1, which is found in
human placenta (10). The two isoforms appear to have similar functional
and pharmacological properties (10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Apparatus for rapid quenching of cells.
a, a glass vessel containing 200 µl of the cell suspension
(+ caged-NO) is situated within the port of a perspex module,
surrounded by a 37 °C water jacket. A UV pulse uncages a fixed
amount of NO, initiating sGC activation. After fixed time intervals, 20 µl of 3 M trichloroacetic acid is expelled under pressure
from a pipette attached to a Picospritzer (Pico). Timing is
coordinated by a Master 8 voltage-step generator. To determine mixing
time, India ink (20 µl) is ejected from the pipette into a vessel
containing buffer (200 µl), at time = 0. This results in a
change in potential difference across a photodiode mounted beneath the
vessel, which is measured with an oscilloscope. The mean of 6 such runs
(± S.E.) is illustrated in b.
Because the experimental material was a cell suspension containing
different cell types, it was important in the first instance to
identify the cells generating cGMP in response to NO over the time
scales used for subsequent biochemical measurements. Previous immunocytochemical studies with the cell suspension had shown that the
cGMP accumulations were restricted to a subpopulation of cells, the
astrocytes, regardless of whether the cells were exposed to NO for
10 s or 2 min (11). However, given that certain cells
(e.g. human platelets) exhibit a transient cGMP signal (11, 16), it was important to ascertain whether any other cell types in our
preparation were making a significant contribution with sub-second
exposures to NO. To address this, paraformaldehyde was injected into
the cell suspension (final concentration, 4%) to fix the cells 100 ms
to 1 s after uncaging NO (from 30 µM caged-NO). The
samples were then double-stained for the astrocytic marker, glial
fibrillary acidic protein (GFAP), and for cGMP. In control cells, only
the occasional cell stained for cGMP, and such cells were also
GFAP-positive (Fig. 2, a and
b). After 100 ms of exposure to NO, additional cGMP-positive
cells were present, although the staining was relatively weak. Again,
cGMP immunoreactivity could not be identified in cells that were not
GFAP-positive (Fig. 2, c and d). The same result
applied when the NO exposures were 500 ms (Fig. 2, e and
f) or 1 s (Fig. 2, g and h),
although the cGMP staining became increasingly intense. It is
reasonable to assume, therefore, that no cell types other than
astrocytes were contributing significantly to the measured cGMP
elevations.
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Rate of sGC Activation--
The levels of cGMP were measured in
cells quenched at different times following flash photolysis of a
concentration (30 µM) of caged-NO that was maximally
effective when assessed at 1- or 10-s time points (see below for
concentration-response relationships). The most detailed time course
(Fig. 3a) covered 0-400 ms,
and this included measurements made every 20 ms for the first 200 ms.
Over this period, the rate of cGMP accumulation appeared roughly linear; the first time point at which cGMP was significantly higher than the basal level was 40 ms (p = 0.001 by Student's
t test). No lag in the onset of cGMP accumulation following
liberation of NO could be discerned.
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Kinetics of sGC Desensitization--
Over the first 400 ms of sGC
activity (Fig. 3a), there was no clear deviation from
linearity in the rate of cGMP accumulation, but over a 1-s time course
(Fig. 3b) the rate became noticeably nonlinear, an effect
that became increasingly evident over a 10-s time course (Fig.
3c). Since the experiments were done under conditions where
degradation of cGMP was abolished (see "Experimental Procedures") the progressive fall in the rate of cGMP accumulation reflects the
progressive desensitization of sGC activity. To describe the cGMP
accumulation with time, the data from the different time courses were
combined into a single progress curve. This was rendered feasible
without having to resort to a normalization procedure because of the
small inter-experiment variation in the absolute values of the cGMP
responses obtained using this technique. The combined data could be
adequately fit by a single exponential function, having a time constant
() of 6.9 s for sGC desensitization; this fit is overlaid on
all the data sets in Fig. 3, a-c (solid line).
Whereas the single exponential provided a reasonable approximation of
the time course as a whole, in no experiment did the line pass through
the starting cGMP level (Fig. 3a). In fact, all 9 individual cGMP measurements at this time point fell below the line, indicating a
significant misfit (p < 0.05 by sign test), which
suggests that there may initially be an additional fast component to
desensitization. The inclusion of a second early exponential (68 ms
time constant) provides a fit to the starting data (not shown).
Rate of sGC Deactivation--
Deactivation refers to the cessation
of sGC activity after the agonist is removed. To investigate the rate
of deactivation, NO was liberated (30 µM caged-NO) and
cGMP accumulation allowed to proceed for 400 ms. A high concentration
of Hb (100 µM) was then added to scavenge the NO in the
bathing medium (scavenging would be achieved in well under 1 ms). As a
check, Hb was injected 20 ms before the UV pulse, and the usual cGMP
accumulation was eliminated (data not shown). The inability of Hb to
penetrate the cells means that the protein cannot directly influence
sGC, and so the rate at which cGMP accumulation levels off
(phosphodiesterase activity being inhibited) corresponds to the rate of
deactivation of sGC. Control cells, which received an equivalent
injection of buffer after 400 ms, continued to accumulate cGMP as
normal (Fig. 4, cf. Fig. 3).
On adding Hb, there was a rapid reduction in the rate of cGMP
accumulation such that the first time point examined (50 ms later) was
significantly less than control (p < 0.05 by
Student's t test). By about 600 ms, cGMP was stable (Fig.
4). The overall time course could be fit by an exponential function
corresponding to a deactivation rate constant of 3.7 s1. This must be regarded as a purely
operational description, however, as the starting gradient of the
fitted exponential (at 400 ms) was less than that of the control,
implying that the true kinetics of deactivation is more complex and may
involve an initial fast phase.
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Concentration-Response Curves for NO Activation of Cellular
sGC--
Concentration-response relationships for NO activation of sGC
were assessed using different concentrations of caged-NO and different
periods of exposure to the liberated NO. At the first time point
examined (100 ms after photolysis), cGMP increased in a simple
sigmoidal manner with logarithmic increases in caged-NO concentration,
the EC50 being about 20 µM and the maximum
being reached at 100 µM (Fig.
5, a and b). The
corresponding concentrations of released NO, estimated by comparing the
quantum efficiency of the caged-NO and caged-ATP (see "Experimental
Procedures"), are 47 and 240 nM. When examined after
1 s stimulation, and more so after 10 s stimulation, however,
the concentration-response curves became bell-shaped. Moreover, at both
time points, the maximum response occurred at the lower concentration
of 30 µM (71 nM NO) and the EC50
was, accordingly, left-shifted to about 10 µM (24 nM NO). These changes in the concentration-response curves
with time are what would be predicted for agonist-induced desensitization (17, 18). Such an effect is well illustrated by the
progressive decline in the rate of cGMP accumulation over the interval
100 ms to 1 s relative to the initial rate (0-100 ms) as the NO
concentration is raised (Fig. 5c). Accordingly, the potency
of NO at the earliest time point (100 ms) should correspond to that at
which sGC desensitization exerts the least influence. The variation in
this initial rate of cGMP formation with NO concentration (estimated)
could be adequately fit by a standard Hill plot, which showed an
EC50 for NO of 45 nM (Fig. 5c,
inset).
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To examine quantitatively how the rate of desensitization is influenced
by agonist concentration, the time course of cGMP accumulation over
10 s at a high concentration of caged-NO (300 µM;
710 nM NO) was examined in more detail. As predicted, the rate of cGMP accumulation at the higher concentration was slower, indicating an enhanced rate of desensitization (Fig. 5d). An
exponential fit to the data indicated a shortening of the time constant
for desensitization from 6.9 to 5.4 s.
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DISCUSSION |
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The development of a new method for rapid quenching of cell suspensions has enabled cGMP responses in cerebellar cells to be satisfactorily resolved on a sub-second time scale. The major factor limiting the time resolution of the method is the mixing time (5 ms). In principle, the approach can be applied to any cell type that can be kept in suspension, although it could be adapted easily for studying cells adhering to a solid surface, e.g. cells cultured on coverslips. Rapid changes in the levels of a variety of metabolites, second messengers, etc. could be measured. The apparatus is fairly inexpensive and relatively simple to construct and use, and it allows highly reproducible results from one day to another. Initially, attempts were made to use a classical, commercially available, quench flow device, as used previously for measurements of cyclic nucleotides in olfactory cilia (19). However, in practice, the quench flow apparatus was found to have several disadvantages. For example, the time ranges permissible within a given experiment are limited, relatively large volumes of cell suspensions are needed, and the cells tend to settle out within the apparatus, causing variation in the numbers sampled. Furthermore, whereas small structures such as olfactory cilia may survive well, the forcing of whole cells under pressure through narrow-bore tubing creates potential problems associated with shear stresses (which vary in magnitude and duration with reaction time). With the present technique, the absolute values for cGMP in cells exposed to NO are closely similar to those reported previously from the use of simpler methods (11), suggesting the new method does not compromise the functional properties of the cells.
The primary aim was to obtain quantitative information on the kinetics
of sGC activation, deactivation, and desensitization, and the results
are summarized in schematic form in Fig.
6a. A model analogous to ones
developed to describe the binding and gating of neurotransmitter
receptors (20) is depicted in Fig. 6b.
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The activation kinetics (predicted to be second order) was too rapid to
be resolved even with a 20-ms sampling time (at 30 µM
caged-NO). No delay between uncaging of NO and the start of cGMP
synthesis was detectable, although the data could accommodate a lag of
a few milliseconds. This lack of detectable delay also applied to lower
NO concentrations (see Fig. 5c). From studies of purified
sGC (21-23), there appears to be general agreement that the NO-free
state of sGC heme is a 5-coordinate ferrous (Fe2+) center,
with a proximal bond to the histidine residue at position 105 of the
-subunit. Binding of NO, creating the 6-coordinate form of the
enzyme, is extremely rapid (near diffusion limited) and is followed by
a transition to the catalytically active 5-coordinate species as a
result of cleavage of the histidine bond (Fig. 6b). This
step is considered to be rate-limiting. One report has concluded that
the 6- to 5-coordinate transition is NO
concentration-dependent in that at sub-stoichiometric
levels of NO there is a pronounced lag in onset of catalytic activity
(at 4 °C), which decreases at higher levels of NO (22). This has
been interpreted as evidence for a second (nonheme) NO-binding site.
With NO in excess over sGC, the lag was around 100 ms, and this would
presumably be much less at physiological temperatures. Another report
(21) indicated that the transition occurred exponentially with a rate
constant of 38 s
1 (half-time = 18 ms) at
15 °C. One assumes that the half-time would reduce to a few
milliseconds at 37 °C. Consequently, there is no indication from our
data that the kinetics of activation of sGC determined using the
purified enzyme in an artificial environment is not applicable to the
enzyme inside cells.
The rate of deactivation, however, represents one parameter where there
is a major divergence between the behavior of purified and cellular
sGC. For reasons that are unclear, there is disagreement between
different laboratories on the rate of deactivation of the purified
enzyme. Kharitonov et al. (24) using Hb or myoglobin to
remove NO estimated the half-life for loss of NO from nitrosyl-sGC was
2 min at 37 °C. When substrate (GTP) and cofactor (Mg2+)
were included, however, the half-life was greatly accelerated to an
estimated 5 s at 37 °C. Brandish et al. (25), in
contrast, obtained a 2.5-3-min half-life (at 37 °C) irrespective of
the presence of GTP and Mg2+. Margulis and Sitaramayya
(26), on the other hand, reported from sGC activity measurements (with
GTP and Mg2+ present) a deactivation half-time of 18 s
(20 °C), extrapolated to 5 s at 37 °C. Our experiments
indicate that sGC deactivation in cells at 37 °C is much faster and
does not occur in a kinetically straightforward manner. The data could
be described operationally as an exponential decline, with a rate
constant of 3.7 s1. Strictly speaking, this
constant describes the rate at which sGC catalytic activity declines to
zero, and this may not just reflect deactivation if progressive
desensitization also contributes to the decline in sGC activity. In
this case, the rate constant for deactivation would be the measured
rate constant (k = 3.7 s
1)
minus the rate constant for desensitization (k = 0.14 s
1) over the 600-ms period, giving a slightly
lower value of 3.56 s
1. This correction,
however, makes the assumption that desensitization would proceed as
normal after removal of free NO. As NO appears to contribute to
desensitization (see below), this assumption may not be valid.
Consequently, we favor an operational constant of 3.7 s
1 as a description of the slowest rate at
which sGC deactivates after removal of NO. This corresponds to a
half-time of 190 ms, a value 25-fold faster than the fastest estimate
made using purified sGC (24, 26).
Mechanistically, it is unclear exactly what the value is describing. In
the model (Fig. 6b), the rate of deactivation would correspond to the rates of the "back" reactions for NO dissociation from sGC (i.e. the steps with the constants
k1 and k
2) minus the
rates of the "forward" reactions (k1 and
k2), and the observed rate constant would
correspond to the rate-limiting step; this is likely to be the
transition from the active, 5-coordinated species to the 6-coordinated
species (k
2; Ref. 22). Alternatively (or in
addition), NO could dissociate directly from the 5-coordinate state
without passing through the 6-coordinate intermediate, and the heme
would then presumably revert to the resting, 5-coordinate, histidine-bound form; if so, this is to what the rate constant would
correspond. Either way, the results imply that factors exist within
cells that enable sGC to deactivate much faster than has so far been
observed for the purified enzyme.
Desensitization of sGC has not been observed with the purified enzyme but was first identified in a previous study on cellular sGC (11). In the present investigation, the falling off of sGC activity with time could be adequately described with a single exponential. A desensitization time constant of 6.9 s was found when the NO concentration was one giving maximal sGC activity after 1 or 10 s, but at a 10-fold higher concentration, the value decreased, indicating that NO accelerates desensitization. There was a hint from the earliest time points that a much more rapid phase of desensitization also exists, but realistically, different methods are needed to probe these very early kinetics accurately. At present, very little is known of the mechanism of desensitization, and in principle, there are several possible routes to the desensitized state of sGC (Fig. 6b). It has to be assumed that cells are equipped with a factor that interacts with sGC to bring about this modification of activity. Two of the simpler hypotheses are as follows: (a) that the factor(s) compromises the catalysis of cGMP synthesis without interfering with NO binding and sGC activation; (b) that the factor interferes with the mechanism of NO binding, dissociation, or enzyme activation, thereby "locking" sGC in a low activity state.
One consequence of the acceleration of sGC desensitization by NO is that the concentration-response curve becomes bell-shaped with time. At the shortest exposure examined (100 ms), when sGC will be close to nondesensitized, the apparent EC50 for NO was about 45 nM. This is 5-fold lower than the EC50 value estimated for the nondesensitizing purified enzyme (27) but is similar to the value measurable from other published data (28, 29). With the onset of desensitization (1 or 10 s of exposure), however, the apparent EC50 fell to about 20 nM. For the 10-s time point, this value should be considered an upper limit because the half-life of NO in the cell suspension is 10 s or less.2 The reduction in EC50 with time may, in part at least, simply reflect the maximal rate of activation becoming truncated by desensitization (cf. Fig. 5b), although it is also possible that the desensitized form has a higher affinity for NO or that the efficacy for transition to the desensitized state is greater than for transition to the nondesensitized state. A method for maintaining NO at a constant concentration will be needed to understand the kinetics of desensitization in further detail.
Functionally, the kinetic properties of sGC will play an important role in decoding NO signals. First, the minimal delay between the arrival of NO and the generation of cGMP (at most a few ms) means that sGC should be capable of registering transient pulses of NO almost contemporaneously with their generation. In neurons, NO synthase is activated by Ca2+/calmodulin, typically as a result of Ca2+ influx through ion channels associated with the N-methyl-D-aspartate subtype of glutamate receptor, to which the NO synthase is tethered via postsynaptic density proteins (2). Following release of a single quantum of glutamate, N-methyl-D-aspartate receptor channel opening leads to an elevation of cytosolic Ca2+ lasting for periods in the 100-ms range (30), and so a minimal NO pulse should have a similar duration. Such a pulse, over the same time scale, should be able to diffuse 10s of micrometers (31) to access sGC in neighboring cells and then activate the enzyme with negligible delay. The kinetic matching between NO generation and the activation of sGC located at a distance should therefore provide the receptive cell with temporal information about NO generation.
The rate of deactivation of sGC when the NO signal disappears is another key parameter governing the dynamics of the signaling pathway, and again, our finding that the enzyme becomes inactive in the 100-ms time scale indicates a kinetic fit to other properties of the pathway, although the dynamics of NO itself in vivo are still unclear. Hence, sGC activity is unlikely to retain a significant "memory" for NO as would be the case if deactivation took the several minutes suggested by some of the data on the purified enzyme (25).
Finally, during more prolonged periods of NO formation the transition,
over a time scale of seconds, of sGC to its desensitized state may
serve two functions. First, it would conserve GTP once time has been
allowed for the cGMP concentration to rise. An analogy here is the way
one heats up soup, using full heat to begin with and then turning it
down as the desired temperature is approached. Second, in cells with
different phosphodiesterase activities, desensitization permits diverse
patterns of cGMP responses, ranging from brief transients to prolonged
plateaus (11). Given the limited molecular heterogeneity of sGC and NO
synthase, this may be a major way that diversity is introduced into the
NO signaling pathway.
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ACKNOWLEDGEMENTS |
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We thank Andrew Batchelor for help in the design of the rapid quenching apparatus, David Goodwin for assistance with immunocytochemistry, and John Wood (SmithKline Beecham, Harlow, UK) for expert advice on data analysis. We also thank Ingo Weyand, Johannes Solzin, and U. Benjamin Kaupp (IBI, Forschungzentrum Juelich, Germany) for advice and assistance in assessing traditional quench-flow methods.
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FOOTNOTES |
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* This work was supported by a Medical Research Council studentship (to T. C. B.), European Community Grant B104-CT98-0034, and The Wellcome Trust.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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 00 44 20 7679 6694; Fax: 00 44 20 7209 0470; Email: john.garthwaite@ucl.ac.uk.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M006677200
2 C. H. Griffiths and J. Garthwaite, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: NO, nitric oxide; sGC, soluble guanylyl cyclase; caged-NO, potassium ruthenium nitrosyl pentachloride; caged-ATP, ATP, P31,-(2-nitrophenyl)ethyl ester (disodium salt); GFAP, glial fibrillary acidic protein; DAPI, 4',6-diamidino-2-phenylindole; P.D., potential difference.
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