From the University Department Of Pharmacology, Oxford University
Oxford OX1 3QT, United Kingdom and School of Pharmacy and
Pharmacology, University of Bath, Claverton Down,
Bath BA2 7AY, United Kingdom
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ABSTRACT |
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Oscillations of Ca2+ in heart
cells are a major underlying cause of important cardiac arrhythmias,
and it is known that Ca2+-induced release of
Ca2+ from intracellular stores (the sarcoplasmic reticulum)
is fundamental to the generation of such oscillations. There is now
evidence that cADP-ribose may be an endogenous regulator of the
Ca2+ release channel of the sarcoplasmic reticulum (the
ryanodine receptor), raising the possibility that cADP-ribose may
influence arrhythmogenic mechanisms in the heart. 8-Amino-cADP-ribose,
an antagonist of cADP-ribose, suppressed oscillatory activity
associated with overloading of intracellular Ca2+ stores in
cardiac myocytes exposed to high doses of the The release of Ca2+ from the sarcoplasmic reticulum
(SR),1 mediated by
Ca2+ release channels known as ryanodine receptors (RyRs),
is believed to play an important role not only during normal cardiac
muscle contraction but also during abnormal conditions associated with Ca2+ overload and oscillations of cell Ca2+ and
membrane potential (1). These oscillations of intracellular Ca2+ are thought to arise from cyclical release and
reuptake of Ca2+ by the SR stores and have been suggested
to underlie a variety of disturbances of the rhythm of the heart
(cardiac arrhythmias).
Ryanodine, which is known to interfere with the function of the SR by
an action at RyRs, suppresses oscillations of Ca2+ in
cardiac myocytes, demonstrating the important role of the RyR and
Ca2+-induced Ca2+ release (CICR) in sustaining
spontaneous activity (2). In recent years, cADP-ribose (cADPR)
has emerged as a possible endogenous regulator of RyR function by
enhancing the sensitivity of CICR to Ca2+ (3-6). Enzymes
for the synthesis and breakdown of cADPR are present in cardiac muscle
(7-10), and endogenous levels have been estimated to be approximately
200 nM (11). Studies in intact heart cells have yielded
results consistent with a role for endogenous cADPR in the regulation
of excitation-contraction coupling. In guinea pig cardiac myocytes
stimulated to fire action potentials, Ca2+ transients and
contractions are enhanced by intracellular applications of cADPR (12)
and reduced by antagonists of cADPR, 8-amino-cADPR and 8-bromo-cADPR
(12-17). These observations are consistent with endogenous cADPR,
acting to enhance the Ca2+ sensitivity of CICR, an action
that may be antagonized by 8-amino-cADPR and 8-bromo-cADPR.
In this study we have investigated (in guinea pig ventricular cells)
the possible influence of 8-amino-cADPR and 8-bromo-cADPR on
spontaneous release of Ca2+ from the SR under conditions of
Ca2+ overload. The two maneuvers chosen to induce
Ca2+ oscillations involved the use of isoproterenol and
ouabain to generate Ca2+ overload. In addition, we have
examined whether exogenous application of cADPR may itself be
arrhythmogenic under certain conditions.
Cell Isolation--
Myocytes were isolated enzymatically from
guinea pig ventricle as described previously (18, 19) and superfused at
34-36 °C with a solution containing 118.5 mM NaCl, 14.5 mM NaHCO3, 4.2 mM KCl, 1.18 mM KH2PO4, 1.18 mM
MgSO4·7H2O, 2.5 mM
CaCl2, 11.1 mM glucose (oxygenated at 95%
O2, 5% CO2).
Electrophysiology--
In experiments investigating the
actions of 8-amino-cADPR, cells were impaled with double-barreled
"theta glass" sharp microelectrodes, allowing one barrel
(containing 1 M potassium methyl sulfate + 10 mM KCl) to be used for electrical recording and injection
of current and the other for cytosolic application of drugs (after obtaining a series of measurements in the absence of drug). Drugs were
applied to the cell during the course of an experiment by injecting a
small quantity of the appropriate solution into the previously empty
barrel of the electrode; this traveled to the tip of the electrode by
capillary action and entered the cell by diffusion. In experiments
investigating the actions of cADPR, electrical recordings were made
from cells using conventional whole-cell (ruptured patch) patch clamp
techniques. When drugs were applied to the cytosol, these were included
in the pipette solution. Control records were taken in the first min,
when it was believed that dialysis from the pipette to the cytosol was minimal, and compared with records 5 to 10 min after rupture of the
membrane, when access of the compound to the cytosol was thought to be
well established. An Axoclamp 2A recording system was used for switched
voltage clamp.
Generation of Oscillatory Activity with
Isoproterenol--
Spontaneous contractile and electrical activity was
induced by exposure of cells to isoproterenol (20-100 nM,
applied continuously). In preliminary experiments, it was found that
continuous superfusion with 20 nM isoproterenol provoked
spontaneous activity that was stable over a period of at least 8 min.
Isoproterenol-induced spontaneous activity was monitored both in
cells stimulated to fire action potentials and (in a separate series of
experiments) under voltage clamp conditions (with 200-ms step
depolarizations from a holding potential of Generation of Oscillatory Activity with Ouabain--
Spontaneous
contractile and electrical activity was induced by exposure of cells to
ouabain (1 µM, applied for 6-10 min and then washed
away). Continuous superfusion with 1 µM ouabain
throughout the course of an experiment did not produce stable
oscillatory behavior but caused the generation of oscillations, which
increased in severity with time, resulting in the gradual deterioration of the cell; however, ouabain-provoked oscillations, once induced, were
found to be stable for a period exceeding 8 min after washout of
ouabain. For this reason, in experiments investigating the effects of
drugs on ouabain-induced oscillations, superfusion with ouabain was
discontinued before cytosolic injection of drugs (injection of drugs
was carried out within 1 min of washout of ouabain). Ouabain-induced
spontaneous activity (in cells not loaded with fura-2) was monitored
using a voltage clamp protocol of trains of 5 200-ms step
depolarizations (from Intracellular Free Ca2+ Measurements Using
Fura-2--
Fura-2 fluorescence was monitored from cells preincubated
with the acetoxymethyl ester of fura-2 (5 µM) for 15-20
min; after loading, a period of at least 30 min was allowed before
experimentation for deesterification of the intracellularly accumulated
fura-2-AM. Excitation light (wavelength 340 ± 5 or 380 ± 5 nm) from a xenon arc lamp (75 W) was delivered by means of a fiberoptic
(diameter 125 µm) with enhanced ultraviolet transmission positioned
very close to the cell under study; emitted fluorescence light
(500 ± 40 nm) was collected through the microscope objective and
quantified by means of a photomultiplier tube.
Imaging of Ca2+ Waves Using Fluo-3--
Fluo-3
fluorescence was monitored from cells preincubated with the
acetoxymethyl ester of fluo-3 (5 µM) for 15-20 min;
after loading, a period of at least 30 min was allowed before
experimentation for deesterification of the intracellularly accumulated
fluo-3-AM. Excitation light (wavelength 485 ± 11 nm) from a xenon
arc lamp (75 W) was delivered by means of a fiberoptic (diameter 125 µm) with enhanced ultraviolet transmission positioned very close to the cell under study; emitted fluorescence light (530 ± 15 nm) was collected through the microscope objective and captured with a
Photonic Science Isis III-intensified CCD camera.
Confocal Microscopy--
Myocytes imaged under the confocal
microscope were loaded with fluo-3 as described above. A Leica TCS NT
confocal scanning head was coupled to a DMIRB microscope with a 63×
water immersion objective lens. Illumination was provided by a 488-nm
Ar laser, and a 515-nm long pass filter was used in the collection of
emitted fluorescence. Line scan imaging was used to maximize temporal resolution; a single line along the long axis of the heart cell was
repeatedly scanned at an acquisition rate of 385 Hz, and images were
constructed by displaying successive lines (corresponding to 66 µm in
length) adjacent to each other.
Statistics--
Values are expressed as mean ±S.E.
Although low doses of isoproterenol (5 nM) cause an
approximate doubling of Ca2+ currents and contractions in
guinea pig ventricular cells with no initiation of spontaneous
activity,2 high doses (20 nM and higher) provoke spontaneous electrical activity.
Fig. 1 illustrates spontaneous electrical
activity recorded from a representative cell exposed to 20 nM isoproterenol and stimulated to fire action potentials
at a frequency of 1 Hz. In the absence of any drugs (Fig.
1A), only stimulated action potentials were recorded, and no
spontaneous electrical activity was observed. However, subsequent
exposure to isoproterenol resulted initially (1-2 min) in the
appearance of small transient depolarizations between stimulated action
potentials (after-depolarizations), which increased in magnitude until
sufficiently large to elicit spontaneous action potentials (each
associated with a spontaneous contraction). Examples of spontaneous
action potentials recorded in this cell after a 3-min superfusion with
isoproterenol are shown in Fig. 1B. Fig. 1C shows
the effect of cytosolic infusion of 8-amino-cADPR (pipette
concentration of 20 µM, dissolved in 20 mM
HEPES buffer) in the continued presence of isoproterenol. Within 3 min
of 8-amino-cADPR injection, spontaneous electrical activity was
completely abolished in this cell, whereas the generation of stimulated
action potentials was not affected. Similar experiments were carried
out in a total of 8 cells. In 6 of these, isoproterenol-induced spontaneous electrical activity was completely (4 cells) or partially (2 cells) inhibited by 8-amino-cADPR within 5 min. No apparent influence of 8-amino-cADPR was observed in the remaining 2 cells. Overall, the number of spontaneous events recorded over a 5-s period
was significantly reduced from 6.4 ± 0.7 to 2.8 ± 1.2 (p < 0.05; n = 8) within 5 min of
8-amino-cADPR infusion.
-adrenoreceptor agonist isoproterenol or the Na+/K+-ATPase
inhibitor ouabain. The oscillations suppressed by 8-amino-cADP-ribose included intracellular Ca2+ waves, spontaneous action
potentials, after-depolarizations, and transient inward currents.
Another antagonist of cADP-ribose, 8-bromo-cADP-ribose, was also
effective in suppressing isoproterenol-induced oscillatory activity.
Furthermore, in the presence of ouabain under conditions in which there
was no arrhythmogenesis, exogenous cADP-ribose was found to be capable
of triggering spontaneous contractile and electrical activity. Because
enzymatic machinery for regulating the cytosolic cADP-ribose
concentration is present within the cell, we propose that
8-amino-cADP-ribose and 8-bromo-cADP-ribose suppress cytosolic
Ca2+ oscillations by antagonism of endogenous cADP-ribose,
which sensitizes the Ca2+ release channels of the
sarcoplasmic reticulum to Ca2+.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 mV to +40 mV at a
frequency of 1 Hz). In a further series of experiments, oscillations of
intracellular Ca2+ induced by isoproterenol were monitored
directly from the fluorescence of fura-2. Cells loaded with fura-2 were
found to be less susceptible to the arrhythmogenic actions of
isoproterenol, possibly because of the Ca2+-buffering
action of the Ca2+ indicator, and hence different
conditions were employed from those used to study myocytes not loaded
with the Ca2+ dye. Cells were superfused with 120 nM isoproterenol, and the voltage clamp protocol consisted
of trains of 20 200-ms step depolarizations from
70 mV to +40 mV at a
frequency of 3.3 Hz, with a 5-s interval between trains. With these
protocols, no spontaneous activity was recorded in the absence of isoproterenol.
70 mV to +40 mV) at a frequency of 2.5 Hz, with
a 7-s interval between trains. With these protocols, no spontaneous
activity was observed in the absence of ouabain.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of 8-amino-cADPR on spontaneous
action potentials provoked by isoproterenol. A,
intracellular records of membrane potential recorded from a ventricular
myocyte isolated from guinea pig heart. Action potentials were evoked
by electric current stimuli (2-ms duration) applied at the
arrows. B, records in the same cell as for
panel A showing spontaneous action potentials provoked by
application of ( )-isoproterenol (20 nM). Note the lack of
correlation, with arrows marking current stimuli.
C, suppression of the spontaneous activity (with action
potentials occurring only at the time of the current stimuli applied at
the arrows) by 8-amino-cADPR (20 µM) dissolved
in HEPES buffer (20 mM) and applied to the cytosol through
one barrel of a microelectrode made using theta glass tubing.
D, action potentials recorded in another cell under the same
conditions as for panel A. E, spontaneous activity recorded
in the same cell shown in panel D following application of
isoproterenol (i.e. same conditions as for panel
B). F, activity in the same cell as for panels
D and E recorded 5 min after cytosolic application of
HEPES buffer without 8-amino-cADPR. Note that spontaneous activity
persisted under these conditions.
In the cells in which 8-amino-cADPR suppressed spontaneous activity, these actions appeared to develop progressively with time; spontaneous action potentials were superceded by after-depolarizations, and after-depolarizations appeared to gradually decrease in magnitude and occur with progressively increasing delay after the preceding stimulated action potential. Such a progressive suppression of oscillatory activity may reflect a gradual increase of 8-amino-cADPR concentration within the cell.
As a control for the above series of experiments, the effects of injection of HEPES buffer (20 mM) in the absence of 8-amino-cADPR were investigated. In 6 of 9 cells studied, little or no effect of HEPES was observed on isoproterenol-induced spontaneous electrical activity (data from a representative cell are shown in Fig. 1, D, E, and F). In 2 cells, a slight increase in the frequency of occurrence of spontaneous activity was observed, whereas in 1 cell, a small decrease was noted. Overall, HEPES was without significant effect; the number of spontaneous events recorded before and 5 min after injection of HEPES was 6.4 ± 0.6 and 6.9 ± 0.7, respectively (p > 0.05; n = 9).
In a further series of experiments, the effects of 8-amino-cADPR and
HEPES on isoproterenol-induced oscillations were investigated under
voltage clamp conditions to avoid any effects that might arise from
changes in action potential duration. Cells were stimulated at a
frequency of 1 Hz with 200-ms step depolarizations from a holding
potential of 70 mV to +40 mV. In all cells studied, superfusion with
20-100 nM isoproterenol for 3 min resulted in the
appearance of transient inward currents between depolarizing pulses; a
representative example is presented in Fig.
2. These transient inward currents are
likely to be secondary to spontaneous Ca2+ release from an
overloaded SR and are thought to be carried predominantly by
sarcolemmal Na+/Ca2+ exchange operating in the
Ca2+ extrusion mode (20, 21). In this cell, subsequent
infusion of 20 µM 8-amino-cADPR (in the continued
presence of isoproterenol) was associated with complete suppression of
transient inward currents within 5 min (Fig. 2B). In a total
of 9 cells investigated, 5 showed complete suppression of transient
inward currents within 5 min of 8-amino-cADPR infusion; in 2 cells, the
magnitudes of transient inward currents were reduced, whereas in a
further 2 cells, there was little or no change. Overall, 8-amino-cADPR
(5 min) suppressed the frequency of occurrence of transient inward currents from 0.84 ± 0.08/step depolarization to 0.33 ± 0.07 (p < 0.05; n = 9) and reduced the
magnitude of the first transient inward current to 42 ± 7%,
that of the preinjection value (p < 0.05;
n = 9).
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In contrast, infusion of HEPES did not reduce the frequency of occurrence and magnitudes of isoproterenol-induced transient inward currents in 5 cells studied. Results from a representative cell are presented in Fig. 2, C and D.
To monitor more directly isoproterenol-induced Ca2+
oscillations, experiments similar to those described above were
repeated in cells loaded with the fluorescent Ca2+
indicator fura-2, using a photomultiplier tube to collect emitted fluorescence. It was found that cells loaded with fura-2 were not as
susceptible to exhibit spontaneous oscillations in cytosolic Ca2+, possibly because of the buffering action of the
Ca2+ indicator, and hence more vigorous conditions were
required to generate oscillatory changes in Ca2+ (see the
Fig. 3 legend). In 5 of 7 cells studied,
complete cessation of isoproterenol-provoked Ca2+
oscillations occurred within 6 min of 8-amino-cADPR injection (20 µM) despite continuous superfusion with isoproterenol.
Data from a typical cell are presented in Fig. 3. In the absence of isoproterenol, the fluorescence signal was stable between trains of
voltage clamp pulses; in the presence of isoproterenol, transient deflections in the fluorescence signal were recorded between trains of
voltage clamp pulses, representing spontaneous elevations of intracellular Ca2+ (Fig. 3A). These oscillations
of cytosolic Ca2+ were abolished within 6 min of
8-amino-cADPR infusion (Fig. 3B). In the remaining 2 cells,
8-amino-cADPR infusion was associated with prolongation of the time to
onset of the spontaneous event, with little change in the magnitude. In
these 7 cells, injection of 8-amino-cADPR was associated with
significant (p < 0.05) reductions both in the number
of spontaneous oscillations per train of voltage clamp pulses (from
1.14 ± 0.14 to 0.29 ± 0.18) and in the peak magnitude of
the first oscillation following a train of pulses, as determined from
the fura-2 fluorescence signal (to 27 ± 18% that before
8-amino-cADPR injection). In six control experiments, HEPES was not
effective in suppressing isoproterenol-induced oscillations under these
conditions (data from a representative cell are shown in Fig. 3,
C and D).
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In another series of experiments, isoproterenol-provoked spontaneous
Ca2+ waves propagating across the cell were recorded using
an image-intensified CCD camera. An example of a spontaneous
Ca2+ wave, initiated after superfusion with isoprenaline,
is shown in Fig. 4A; two waves
begin near the center of the cell and propagate toward the edges in
opposite directions. In this cell, cytosolic application of
8-amino-cADPR was associated with complete abolition of
Ca2+ waves within 5 min (Fig. 4B). In 12 of 17 cells studied in this way, Ca2+ waves were abolished or
markedly suppressed by 8-amino-cADPR (20 µM) within 8 min.
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The spatial characteristics of spontaneous activity and its suppression
by 8-amino-cADPR were studied in more detail using a laser-scanning
confocal microscope operating in line-scan mode (cells loaded with the
Ca2+ indicator fluo-3). In this configuration, the temporal
resolution was maximized by repeatedly scanning at a rate of 2.6 ms/scan along a single line (66 µm in length) across the long axis of the heart cell. The lower panel of Fig.
5A illustrates the images obtained by this method. Each image (numbered 1-12) is composed of 512 sequential line scans with each line displayed in a vertical orientation. Thus, the horizontal direction in the image represents increasing time (2.6 ms/line, 1331 ms for each image), and the vertical
direction represents the distance along the line scan (66 µm for each
image).
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In this series of experiments, myocytes were repeatedly stimulated to fire trains of 10 action potentials (at 1 Hz) with a 10-s interval between trains and were provoked to exhibit spontaneous Ca2+ waves by superfusion with 50 nM isoproterenol. The upper panel of Fig. 5A shows data from a representative cell and illustrates 10 stimulated action potentials (stimuli applied at blue line) followed by an after-depolarization (arrow) that developed following superfusion with isoproterenol. The lower panel presents the line scan images obtained over this period; dark colors represent low cytosolic Ca2+, whereas red represents high Ca2+. Images 1-6 illustrate elevations in intracellular Ca2+ accompanying each action potential, and it can be seen that the rise in Ca2+ is relatively uniform along the line scan, representing a synchronous global increase in Ca2+. In contrast, image 8 shows a spontaneous Ca2+ wave associated with the after-depolarization; it can be seen that the rise in Ca2+ is not uniform along the line but commences at a point near the lower end of the image before propagating along the length of the cell as time proceeds. Fig. 5B shows data from the same cell 5 min after injection of 20 µM 8-amino-cADPR. 8-Amino-cADPR completely suppressed the generation of Ca2+ waves and after-depolarizations in this cell without abolishing stimulated action potentials or the calcium transients that accompany them. There was also an apparent reduction in the magnitude of the calcium elevations accompanying each action potential; this might be expected, as 8-amino-cADPR has previously been shown to reduce the magnitude of the calcium transient in guinea pig ventricular myocytes (13). However, a contribution of dye loss to the reduction in fluorescence intensity is likely also to contribute.
Data from another cell are shown in Fig.
6. In this cell, isoproterenol-provoked
oscillations were more severe, consisting of spontaneous action
potentials (Fig. 6A, upper panel). The line scan
images (lower panel) reveal that each spontaneous action potential was preceded by a calcium wave that commenced at a point near
the center of the scanned line before propagating bidirectionally toward the edges of the cell. Subsequently a global increase in Ca2+ occurred along the line as the spontaneous action
potential was initiated. Image 3 of Fig. 6A has been
enlarged for clarity: the white arrow indicates a
Ca2+ wave preceding a spontaneous action potential. It is
interesting to note that the global rise in Ca2+
accompanying the spontaneous action potential was not uniform along the
scanned line; instead, the region of the cell to which the wave had
previously propagated showed a smaller increase in Ca2+
than adjacent areas. This may be because of selective depletion of
intracellular Ca2+ stores in this region of the cell or
inactivation of the RyRs involved in the propagation of the wave. After
injection of 8-amino-cADPR, the generation of spontaneous action
potentials was completely suppressed, as shown in Fig. 6B,
although some oscillatory activity persisted in the form of a single
after-depolarization (arrow) accompanied by a
Ca2+ wave (image 3, enlarged for clarity). In
total, 8 cells were imaged in this way; in 6 of these, 20 µM 8-amino-cADPR completely or partially suppressed
isoproterenol-induced calcium waves within 8 min of injection.
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The actions of 8-amino-cADPR were also investigated using another method to provoke Ca2+ oscillations. In this series of experiments, spontaneous activity was initiated by exposure to the cardiac glycoside ouabain rather than isoproterenol. Ouabain has been employed in a number of studies as an experimental tool for the generation of spontaneous Ca2+ oscillations in heart cells (22, 23). Its major mechanism of action is believed to involve binding to the K+ binding site of the sarcolemmal Na+/K+-ATPase and inhibition of its activity, resulting in a secondary rise in the level of intracellular Na+ (24-27). This in turn is thought to inhibit the extrusion of Ca2+ via sarcolemmal Na+/Ca2+ exchange, leading to an elevation of cytosolic Ca2+ and a secondary increase in the quantity of Ca2+ stored in the SR. The spontaneous oscillations of intracellular Ca2+ associated with ouabain toxicity are believed to be because of overloading of the SR with Ca2+; indeed, elevated levels of "luminal" Ca2+ have been reported to enhance the open probability of RyRs studied in planar lipid bilayers (28). Additional mechanisms may also be involved; certain glycosides have been reported to increase Ca2+ entry via sarcolemmal Ca2+ channels (29) and increase the open probability of single RyRs incorporated into artificial lipid bilayers (30). Furthermore, it has recently been reported that ouabain may alter the ion selectivity of sarcolemmal Na+ channels to allow Ca2+ entry ("slip mode conductance"), which might contribute to increased cellular Ca2+ loading (31).
Cytosolic injection of 8-amino-cADPR, but not HEPES, was associated
with a suppression of ouabain-induced transient inward currents;
representative individual experiments are shown in Fig. 7. In 7 cells studied, intracellular
application of 8-amino-cADPR (20 µM) resulted in
reductions both in the number of transient inward currents per train of
voltage clamp pulses (from 1.85 ± 0.48 to 1.00 ± 0.10;
p < 0.05) and in the magnitude of the first transient
inward current following a series of pulses (to 50 ± 6% that
before injection; p < 0.05). In contrast, injection of HEPES (20 mM) was without significant effect on either of
these variables.
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8-Bromo-cADPR is another cADPR analogue that has been shown to act as an antagonist of cADPR, albeit with less potency than 8-amino-cADPR (12, 14). We therefore investigated the actions of this compound in experiments similar to those presented in Fig. 1; cells were stimulated to fire action potentials at 1 Hz, and spontaneous activity was induced by 50 nM isoprenaline. In 6 of 8 cells studied, injection of 100 µM 8-bromo-cADPR was associated with suppression or abolition of spontaneous activity (after-depolarizations and spontaneous action potentials), supporting the hypothesis that this is being achieved through an antagonism of the actions of endogenous cADPR.
Because 8-amino-cADPR and 8-bromo-cADPR, antagonists of cADPR, were
found in the above experiments to be effective in suppressing Ca2+ oscillations under conditions of Ca2+
overload, it was of interest to determine whether, under certain conditions, exogenous cADPR might trigger oscillatory behavior in
previously quiescent cells. In support of this possibility were
previous observations that application of 10 µM cADPR via a patch pipette was found to be associated with the development of
spontaneous activity in a minority of cells (4 of 15). To investigate further whether exogenous cADPR might provoke oscillations, ventricular myocytes were superfused (for 6-7 min) with 1 µM ouabain
and stimulated to fire action potentials at 1 Hz, conditions that alone
do not lead to arrhythmogenesis but which might be expected to increase the tendency for this to occur (through enhancement of SR
Ca2+ loading). Under such conditions, 7 of 9 cells showed
spontaneous contractile and electrical activity within 10 min of
exposure to cADPR (50 µM); a typical example is shown in
Fig. 8. In contrast, omission of cADPR
from the patch pipette in 10 cells superfused with 1 µM
ouabain was not associated with the generation of any oscillatory
behavior.
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DISCUSSION |
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The above data demonstrate a suppressive effect of 8-amino-cADPR and 8-bromo-cADPR on spontaneous Ca2+ waves, contractions, and electrical activity recorded in guinea pig ventricular myocytes under conditions of Ca2+ overload induced by isoproterenol or (in the case of 8-amino-cADPR) ouabain. We have reported previously that, in guinea pig ventricular myocytes stimulated to fire action potentials at 1 Hz, injection of 8-amino-cADPR reduces the Ca2+ transient and accompanying contraction by a mechanism involving a decrease in the quantity of Ca2+ released from the SR but with no reduction in the amount of stored Ca2+ (13). Similarly, application of 8-bromo-cADPR via a patch pipette has also been shown to suppress the Ca2+ transient and twitch contraction of guinea pig ventricular cells (12). The observations presented here are thus consistent with an action of 8-amino-cADPR and 8-bromo-cADPR to inhibit oscillatory activity induced by isoproterenol or ouabain through the suppression of Ca2+ release from the SR, most likely because of effects (either direct or indirect) on the RyR.
In view of the evidence in sea urchin egg preparations and mammalian cells that 8-amino-cADPR and 8-bromo-cADPR are competitive antagonists of cADPR-induced Ca2+ mobilization (14, 17, 32), we propose that suppression of oscillatory activity by these compounds may be because of antagonism of the actions of endogenous cADPR. ADP-ribosyl cyclase activity has been identified in rat cardiac myocytes (7), and intracellular levels of cADPR have been estimated to be of the order of 200 nM (11). Furthermore, cADPR has been reported to enhance CICR in a number of preparations, including guinea pig heart cells (12), sea urchin eggs (33, 34), and neuronal cells (5, 35-37). This would support the hypothesis that suppression of Ca2+ oscillations (provoked by isoproterenol and ouabain) by 8-amino-cADPR and 8-bromo-cADPR is because of antagonism of the actions of endogenous cADPR, which sensitizes the CICR mechanism to Ca2+.
In support of the theory that cADPR amplifies CICR in the heart is the
observation above that, if loading of the SR with Ca2+ was
augmented by exposure to 1 µM ouabain (under conditions
in which this concentration was insufficient to initiate oscillatory behavior alone), exogenous cADPR was associated with the development of
spontaneous contractile and electrical activity. It therefore seems
possible that cADPR may exert an important influence on arrhythmogenic
activity in the heart, particularly under conditions where loading of
the SR with Ca2+ is high, and hence, compounds that reduce
the actions of endogenous cADPR may prove useful in the treatment of
certain cardiac arrhythmias.
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FOOTNOTES |
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* This work was supported by grants from the Wellcome Trust and SmithKline Beecham. Tel.: 44-1865-271613; Fax: 44-1865-271853; E-mail: derek.terrar{at}pharm.ox.ac.uk.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: University Dept. of Pharmacology, Oxford University, Mansfield Rd., Oxford, OX1 3QT UK. Tel.: 44-1865-271613; Fax: 44-1865-271853; E-mail: derek.terrar{at}pharm.ox.ac.uk.
2 S. Rakovic, Y. Cui, S. Iino, A. Galione, G. A. Ashamu, B. V. L. Potter, and D. A. Terrar, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: SR, sarcoplasmic reticulum, RyR, ryanodine receptors; CICR, Ca2+-induced Ca2+ release; cADPR, cADP-ribose.
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REFERENCES |
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