Smooth Muscle Group, Department of Physiology, The Queen's University of Belfast, BT9 7BL Belfast, United Kingdom
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ABSTRACT |
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Freshly dispersed
interstitial cells from the rabbit urethra were studied by using the
perforated-patch technique. When cells were voltage clamped at 60 mV
and exposed to 10 µM norepinephrine (NE) at 80-s intervals, either
large single inward currents or a series of oscillatory inward currents
of diminishing amplitude were evoked. These currents were blocked by
either phentolamine (1 µM) or prazosin (1 µM), suggesting that the
effects of NE were mediated via
1-adrenoceptors.
NE-evoked currents were depressed by the blockers of
Ca2+-activated Cl
currents, niflumic acid (10 µM), and 9-anthracenecarboxylic acid (9-AC, 1 mM). The reversal
potential of the above currents changed in a predictable manner when
the Cl
equilibrium potential was altered, again
suggesting that they were due to activation of a Cl
conductance. NE-evoked currents were decreased by 10 µM cyclopiazonic acid, suggesting that they were dependent on store-released
Ca2+. Inhibition of NE-evoked currents by the phospholipase
C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate
(100 µM) suggested that NE releases Ca2+ via an inositol
1,4,5-trisphosphate (IP3)-dependent mechanism. These
results support the idea that stimulation of
1-adrenoceptors releases Ca2+ from an
IP3-sensitive store, which in turn activates
Ca2+-activated Cl
current in freshly
dispersed interstitial cells of the rabbit urethra. This elevates slow
wave frequency in these cells and may underlie the mechanism
responsible for increased urethral tone during nerve stimulation.
chloride; pacemaker cell; neurotransmission
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INTRODUCTION |
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THE URETHRA IS THOUGHT
to play an important role in maintaining urinary continence by
generating sufficient tone to prevent leakage of urine from the
bladder. Although this tone is largely myogenic in nature, it can also
be augmented by adrenergic nerves acting on postjunctional
1-receptors (6, 7). Recent evidence suggests that Ca2+-activated Cl
channels may
play an important role in the generation of spontaneous myogenic tone
in the urethra (10, 12, 27, 28). However, the precise
mechanisms mediating
1 responses in the urethra remain unknown, although a number of sharp microelectrode recordings made from
the urethras of guinea pigs and rabbits demonstrated that exogenously
applied norepinephrine (NE) could increase the frequency of slow waves
[9, 12].
Recent studies in our laboratory (27, 28) demonstrated that a small population of freshly dispersed cells from the rabbit urethra shared similar characteristics with interstitial cells of Cajal (ICC) believed to be the pacemaker cells in the gastrointestinal (GI) tract (17, 19, 25, 26, 31, 32). These urethral interstitial cells were vimentin positive, noncontractile, and spontaneously active and were therefore postulated to be "pacemaker" cells in the rabbit urethra. A parallel could therefore be made between interstitial cells in the urethra and ICC in the GI tract. Because ICC act not only as pacemakers but also as mediators of neurotransmission (8, 26, 34, 35), it was of interest to investigate whether interstitial cells could play a similar role in the urethra. In support of this hypothesis is the observation that exogenously applied NE increased the frequency of spontaneous transient depolarizations (STDs) recorded from isolated urethral interstitial cells (27), demonstrating that NE could directly influence isolated urethral interstitial (pacemaker) cells. This novel finding may prove to have important implications with regard to mechanisms controlling urethral tone. In this study we have characterized the mechanisms underlying the effect of exogenous NE on isolated interstitial cells and investigated whether these cells contribute to neurogenic responses in the rabbit urethra.
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MATERIALS AND METHODS |
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The bladder and urethra were removed from both male and female rabbits immediately after they had been killed by lethal injection of pentobarbitone. The most proximal 1 cm of the urethra was removed and placed in Krebs solution, and from this, strips were dissected for cell dispersal or for tension recording.
Cell dispersal. Strips of proximal urethra 0.5 cm in width were cut into 1-mm3 pieces and stored in Hanks' Ca2+-free solution for 30 min before being incubated in dispersal medium containing [per 5 ml of Ca2+-free Hanks' solution (see Solutions): 15 mg collagenase (Sigma type 1A), 0.5 mg protease (Sigma type XXIV), 5 mg bovine serum albumin (Sigma), and 15 mg trypsin inhibitor (Sigma)] for 10-15 min at 37°C. Tissue was then transferred to Ca2+-free Hanks' solution and stirred for an additional 15-30 min to release single smooth muscle cells and interstitial cells. These cells were plated in petri dishes containing 100 µM Ca2+ Hanks' solution and stored at 4°C for use within 8 h.
Solutions.
The compositions of the solutions used were as follows (in mM):
1) Hanks' solution, 129.8 Na+, 5.8 K+, 135 Cl, 4.17 HCO
Hanks' solution, 129.8 Na+, 5.8 K+, 86 glutamate, 49 Cl
,
4.17 HCO
, 1.0 Mg2+, 0.5 EGTA, and
10 HEPES, pH adjusted to 7.2 with CsOH; and 4) Krebs
solution 146.2 Na+, 5.9 K+, 133.3 Cl
, 25 HCO
Perforated-patch recordings from single cells.
Currents were recorded by using the perforated-patch configuration of
the whole cell patch-clamp technique (15, 24). This circumvented the problem of current rundown encountered using the
conventional whole cell configuration. The cell membrane was perforated
by using the antibiotic amphotericin B (600 µg/ml). Patch pipettes
were initially front-filled by being dipped into pipette solution and
were then backfilled with the amphotericin B-containing
solution. Pipettes were pulled from borosilicate glass
capillary tubing (1.5-mm outer diameter, 1.17-mm inner diameter; Clark
Medical Instruments) to a tip of diameter ~1-1.5 µm and resistance of 2-4 M.
Isolated tissue recording.
Circularly orientated strips (8 × 1 × 1 mm) of smooth
muscle were removed from rabbit urethra, placed in a water-jacketed organ bath maintained at 37°C, and perfused with warmed Krebs solution that was bubbled with 95% O2-5% CO2.
Strips were adjusted to a tension of 2-4 mN and allowed to
equilibrate for 50 min before experimentation began. Contractions were
measured by using Statham UC3 and Dynamometer UF1 transducers, the
outputs of which were recorded on a Grass 7400 chart recorder.
Transmural nerve stimulation was applied by a Grass S11 stimulator,
which delivered 0.3-ms pulses of 50 V (nominal) at a frequency of 10 Hz. Pulses were applied for 0.1, 0.3, 1, 3, and 10 s. Stimuli were
applied ~1 min after the preceding neurogenic contraction had
returned to baseline levels. All experiments were carried out in the
presence of atropine (1 µM) and
N-nitro-L-arginine (10 µM) to
block contributions from cholinergic and nitrergic nerves.
Drugs.
The following drugs were used: amphotericin B (Sigma),
2-aminoethoxydiphenyl borate (2-APB; Sigma), 9-anthracenecarboxylic acid (9-AC; Sigma),
2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; Sigma),
atropine (BDH Laboratories), caffeine (Sigma), cyclopiazonic acid (CPA;
Calbiochem), dimethyl sulfoxide (DMSO; Sigma),
N-nitro-L-arginine (Sigma),
niflumic acid (Sigma), NE (Levophed; Sanofi Winthrop, UK), penitrem A
(Sigma), phentolamine (Ciba), and prazosin (Tocris).
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RESULTS |
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Digestion of rabbit urethral smooth muscle strips yielded both
interstitial cells and smooth muscle cells. Although the majority of
cells were smooth muscle, interstitial cells could be easily distinguished from smooth muscle cells by using a number of criteria (27). Thus interstitial cells were highly branched, failed
to contract in response to depolarizing current injection or
application of NE, possessed abundant Ca2+-activated
Cl current, and normally fired spontaneous transient
inward currents. In a previous study we demonstrated that NE (10 µM)
enhanced the frequency of slow waves in isolated urethral interstitial
cells, but the mechanisms underlying this effect were not examined
(27). In this study we carried out a series of experiments
to establish the mechanisms through which NE mediates its effects on
these cells.
Effect of NE.
Figure 1 shows typical examples of the
effect of NE (10 µM) on spontaneous activity in interstitial cells
held under voltage clamp. When cells were held at 60 mV, spontaneous
transient inward currents (STICs) were apparent, and application of 10 µM NE evoked a large inward current followed by a series of currents
of diminishing amplitude. These results suggest that the elevation of
STIC frequency underlies the increase in slow wave activity observed
previously (27). The effects of NE were reproducible
within the same cell if a period of 80 s was allowed between
successive applications, presumably reflecting a combination of the
time taken for the NE-sensitive store to refill and the cell to recover
from desensitization. Although the effects of NE on individual
interstitial cells were reproducible, variable responses to NE were
elicited in different cells. In 16 of 35 cells examined, NE evoked
currents similar to those shown in Fig. 1, A and
B. They consisted of a large transient inward current that
was followed by a series of smaller inward currents with a mean
frequency of 7 ± 1 min
1. In the remaining 19 cells,
application of NE evoked large single inward currents similar to that
shown in Fig. 1C.
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Effect of -adrenoceptor antagonists.
Having demonstrated that NE could evoke large transient inward
currents, we assessed the involvement of
-adrenoceptors in this
response by examining the effect of phentolamine and prazosin on the
response to NE. Figure 2A
shows a typical response to NE before, during, and after application of
the nonspecific
-adrenoceptor antagonist phentolamine (1 µM).
Figure 2B shows a summary bar chart for six experiments in
which phentolamine decreased the mean peak inward current from
938 ± 277 pA to
2 ± 2 pA (P < 0.05).
Upon washout, application of NE evoked currents of
816 ± 272 pA. We next examined the effect of the
1-adrenoceptor
antagonist prazosin (1 µM) on the response to NE. Figure
2C demonstrates that the response to NE was mediated via
activation of
1-adrenoceptors because it was reversibly
abolished by prazosin. In five similar experiments, NE evoked inward
currents of
618 ± 278 pA (n = 5) before
application of prazosin. In the presence of prazosin, NE failed to
elicit any inward currents, but upon washout the amplitude of the
NE-evoked current was
669 ± 354 pA.
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Effect of CPA.
To establish whether NE evoked large STICs by releasing
Ca2+ from intracellular stores, we examined the effects of
the Ca2+-ATPase inhibitor CPA (10 µM) on the response.
Figure 3A shows a typical
experiment in which the response to repeated applications of NE was
examined in the presence of CPA. Before application of CPA, NE evoked
large inward currents that became progressively smaller in the presence
of CPA. Figure 3B shows a summary for six similar
experiments in which the peak inward current was reduced from
604 ± 149 pA to
116 pA ± 63 pA after 240 s in CPA
(P < 0.05). Within 160 s of the removal of CPA,
NE evoked large inward currents that were not significantly different
from control (
501 ± 180 pA). These data support the idea that
NE caused the release of Ca2+ from intracellular stores.
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Inhibition of phospholipase C with NCDC.
Previously we demonstrated (21) that the inhibitor of
inositol 1,4,5-trisphosphate (IP3)-dependent release,
2-APB, abolished the response to NE in urethral interstitial cells
(28). More recent studies (23) have
questioned the specificity of 2-APB and suggested that it may act by
inhibiting store refilling rather than inhibiting store release.
Therefore, we examined the effects of the phospholipase C inhibitor
NCDC (100 µM) on NE-evoked currents. Figure
4A shows a typical experiment
in which NE evoked large inward currents before the addition of NCDC.
In its presence, spontaneous activity was abolished as reported
previously (28), and the response to NE was inhibited
(Fig. 4B). Figure 4C shows a summary for six
cells in which NE evoked mean inward currents that were 396 ± 97 pA in amplitude before, compared with
14 ± 9 pA after,
application of NCDC (P < 0.05). To test directly for
the possibility that NCDC blocked Cl
channels, we
examined its effects on Cl
currents evoked by caffeine.
Figure 4D shows a typical experiment in which application of
caffeine (10 mM) to a cell held at
60 mV evoked a large inward
current. In the presence of NCDC (100 µM; Fig. 4E),
application of caffeine evoked a current of similar amplitude to
control. Figure 4F shows a summary of four experiments in
which caffeine application evoked large inward currents of
383 ± 77 pA. In the presence of 100 µM NCDC, the caffeine-evoked currents were slightly increased to
439 ± 67 pA, but this was not significant (P = 0.08). The above data suggest that
the effects of NCDC are not mediated through blockade of
Cl
channels and are consistent with our previous findings
that the effects of NE are mediated by an IP3-dependent
mechanism (28).
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Involvement of Ca2+-activated
Cl channels.
The data presented so far support the idea that activation of
1-adrenoceptors stimulates phospholipase C, leads to the
production of IP3, and consequently releases
Ca2+ from intracellular stores. This, in turn, activates
Ca2+-sensitive channels on the cell membrane and elicits
large transient inward currents. To test whether the NE-induced
currents were carried by Cl
ions through
Ca2+-activated Cl
channels previously
characterized in these cells (27), we examined the
reversal potential of the NE-induced current and the effects of
different Cl
channel blockers on this current. Figure
5A shows the effects of NE on
a cell recorded with symmetrical Cl
solutions [chloride
equilibrium potential (ECl) = 0mV] and
held at a variety of voltages ranging from
60 to +40 mV. When the cell was held at
60 mV, NE application evoked inward currents that
became progressively smaller in amplitude as the cell was depolarized
toward 0 mV. At potentials positive to 0 mV, the currents were outward
and increased in amplitude as the cell was held at more depolarized
potentials. A summary of six similar experiments is shown in Fig.
5B where the peak amplitude of the inward current was
plotted at different holding potentials. Under these conditions, the
NE-evoked current reversed close to 0 mV, suggesting that the current
was carried by Cl
ions. In a separate set of experiments,
we examined the effects altering the external Cl
concentration on the reversal potential of the NE-evoked current. A
series of 400-ms voltage ramps from
50 to +50 mV were applied to
cells every 500 ms before and during application of NE. Figure 5C shows a typical experiment in which the NE-sensitive
currents were obtained and plotted against voltage. Under control
conditions (ECl = 0mV), the NE-sensitive
current reversed at
1 mV, and when the external Cl
concentration was decreased to 49 mM (ECl = 27 mV), the reversal potential of the inward current shifted to 25 mV.
In four similar experiments, the reversal potential of the NE-sensitive
currents shifted from 0 ± 2 mV to 23 ± 2 mV when external
Cl
was reduced from 135 to 49 mM
(P < 0.01, data corrected for junction potentials of
3 mV and +2 mV in normal and reduced external Cl
solutions, respectively).
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Tension recordings.
Having characterized the effects of NE on isolated urethral
interstitial cells, we next wanted to establish whether they played any
role in mediating noradrenergic neurotransmission. To test this
possibility, the effect of transmural nerve stimulation was examined on
strips of urethra in the presence of atropine (1 µM) and
N-nitro-L-arginine (10 µM) to
block the contribution of cholinergic and nonadrenergic noncholinergic
(NANC) transmitters, respectively. Pulses (0.3-ms duration, 10 Hz) were
applied for 0.1, 0.3, 1, 3 and 10 s. Figure 7, A and
C, shows typical responses of a urethral strip to each
stimulation. Increasing the duration of stimulation increased both the
amplitude and duration of contraction. In the presence of phentolamine
(1 µM), the contractile responses to nerve stimulation were
abolished, suggesting that only noradrenergic responses were evoked
under these conditions (n = 3, data not shown). We next
examined the effects of blocking Cl
channels on the
response to nerve stimulation. Figure
7A shows a typical experiment
in which nerve evoked contractions were elicited in the absence and
presence of niflumic acid (100 µM). Because niflumic acid has been
demonstrated to open large conductance Ca2+-activated
K+ (BK) channels (11), all experiments were
carried out in the presence of penitrem A (100 µM) to block BK
channels (14, 18). Application of niflumic acid reduced
spontaneous contractions and decreased the amplitude of nerve-evoked
contractions. The bar chart in Fig. 7B shows a summary of six similar
experiments where the peak contraction amplitude was measured before
and during application of niflumic acid. Data were normalized to the
response obtained by a stimulation duration of 10 s in the absence
of Cl
channel blockers. In the presence of niflumic acid,
the amplitude of contraction was significantly reduced at 0.3, 1, and
10 s to 14 ± 4, 16 ± 5, and 18 ± 4%,
respectively (P < 0.05). The effects of 9-AC (1 mM)
were also examined on neurogenic contractions. Figure 7C
shows a typical response to nerve stimulation before and during
application of 9-AC. Note again that both spontaneous and neurogenic
contractions were depressed in the presence of 9-AC. Figure
7D shows summary data for seven similar experiments in which
9-AC reduced the amplitude of contraction in response to 0.3-, 1-, and
10-s stimulation periods to 16 ± 4, 16 ± 3, and 30 ± 5%, respectively (P < 0.05).
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DISCUSSION |
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A variety of studies have demonstrated that ICC play a central
role in the initiation, coordination, and modulation of spontaneous activity in the GI tract (25, 26, 31). Cells with
characteristics similar to the ICC in the gut have recently been
isolated in a variety of tissues from the lower urinary tract
(22, 27, 28). Although their function in these tissues has
not been thoroughly established, we have hypothesized that urethral
interstitial cells contribute to the generation of tone by acting as
pacemakers to "drive" the surrounding smooth muscle
(27). Two main pieces of evidence support this contention.
First, isolated interstitial cells fire regular spontaneous slow waves,
which resemble the electrical activity recorded in whole tissues with
microelectrodes, whereas smooth muscle cells are electrically quiescent
(12, 13, 27). Second, the slow waves recorded in whole
tissues and isolated interstitial cells are abolished when
Ca2+ release from IP3-sensitive stores is
inhibited or Ca2+-activated Cl channels are
blocked (12, 27, 28)
Modulation of this mechanism by excitatory neurotransmitters such as NE
could provide an efficient means to alter tone, because an increase in
slow wave frequency could lead to an enhancement of contractile force.
Previous studies have demonstrated that NE is the major excitatory
transmitter in the urethra (2, 3), and for this reason we
examined the mechanisms of its action on rabbit urethral interstitial
cells. Hashitani et al. (12) demonstrated that NE
increases the frequency of STDs in the rabbit urethra by enhancing the
oscillatory release of Ca2+ from intracellular stores,
which results in the periodic activation of Cl channels.
Because urethral smooth muscle cells in the rabbit possess little
Ca2+-activated Cl
current (27),
it is unlikely that the observed changes in electrical activity
recorded with intracellular microelectrodes reflect an action on these
cells and leaves the intriguing possibility that interstitial cells act
as the primary target for neuronally released NE.
In a previous study (27), we demonstrated that under
current-clamp conditions, NE produced responses in isolated
interstitial cells that appear to be remarkably similar to those
recorded in whole tissue preparations with sharp microelectrodes
(12). Thus NE produced an initial large slow wave that was
followed by a series of more frequent but shorter slow waves. The
reason for such a pattern of activity became apparent when cells were
held under voltage clamp, where NE typically induced a large inward current that was followed by more frequent, gradually diminishing inward currents. A number of lines of evidence suggest that the NE-evoked currents reflected the stimulation of
Ca2+-activated Cl currents that have been
demonstrated in a variety of smooth muscles (1, 20).
First, when cells were held under voltage clamp, the currents reversed
close to ECl in symmetrical Cl
solutions and shifted in a predictable manner when
ECl was altered. Second, they were significantly
decreased by either niflumic acid or 9-AC. Although the concentration
of 9-AC (1 mM) used to block the response is relatively high compared
with other tissues (20), we have previously demonstrated
that this concentration selectively blocks the Cl
current
in urethral interstitial cells (27).
When we examined the pharmacology of the NE response, it appeared to be
similar to the pathway described in a number of smooth muscles
(1, 4, 5, 30). Thus the response to NE was abolished by
either phentolamine or prazosin, suggesting that it was mediated via
activation of 1-adrenoceptors. Similarly, its effects
were attenuated when phospholipase C was blocked with NCDC, which is
consistent with our previous observation that the IP3
inhibitor 2-APB (21) inhibits NE responses in urethral
interstitial cells (28). These data support the idea that
NE mediates its effects by upregulating the normal pacemaking mechanism
in these cells.
In recent years, a number of studies have demonstrated that ICC in the
gut act as important intermediaries in both excitatory and inhibitory
neurotransmission (8, 26, 34, 35). The evidence to support
this role in the gut has been obtained by using a variety of techniques
including gene knockout technology. Perhaps the most convincing
demonstration that ICC play a central role in neurotransmission in the
gut has been obtained from mutant mice (W/Wv) that failed
to develop intramuscular ICC. In these animals, both excitatory and
inhibitory nerve evoked responses were greatly attenuated (8, 34,
35). Unfortunately, similar rabbit knockouts are presently
unavailable. However, we have previously demonstrated that in marked
contrast to the smooth muscle cells, interstitial cells in the urethra
possess abundant Ca2+-activated Cl current
(27). We exploited this difference in the present study to
assess the contribution of interstitial cells to noradrenergic neurotransmission in strips of urethra by attempting to
pharmacologically knock out interstitial cells using Cl
channel blockers. Although this method lacked the elegance of genetic
knockout technologies, it produced effects that were consistent with
abolishing the contribution of interstitial cells. Thus, in the
presence of either niflumic or 9-AC, both spontaneous and neurogenic
contractions were attenuated. These data tentatively suggest that
interstitial cells in the urethra not only contribute to spontaneous
mechanical activity but also may play an important role in neurotransmission.
Although the idea that interstitial cells mediate neurotransmission in
the urethra may be appealing, the results obtained on whole tissue
strips should be interpreted with caution. This preparation consists of
a complex arrangement of nerves, smooth muscle, and interstitial cells,
all of which may be affected by the Cl channel blockers.
The observed effects on neurogenic contractions may be caused by a
presynaptic action of the Cl
channel blockers, which
could inhibit neurotransmitter output. However, inhibition of neuronal
Cl
channels should prolong neuronal action potentials
and, consequently, enhance neurotransmitter output. Alternatively, the
Cl
channel blockers could inhibit neurotransmitter output
by blocking neuronal Ca2+ channels. Although we cannot rule
out this possibility, it is interesting to note that Jackson et al.
(16) have failed to demonstrate any effect of niflumic
acid on action potential-evoked axonal Ca2+ transients in
the rat vas deferens, suggesting that Ca2+ influx is
unaffected by niflumic acid.
Recent immunohistochemical evidence presented by Smet et al. (29) and Waldeck et al. (33) would also support our contention that urethral interstitial cells play a role in neurotransmission. These studies demonstrated the presence of branched interstitial cells in the human, guinea pig, and rabbit urethras, respectively, that were immunopositive for cGMP and support the idea that they may be important in mediating neurally released nitric oxide responses. Unfortunately, the detailed immunohistochemistry and electronmicroscopy data that have been provided to support the role of ICC as mediators of neurotransmission in the gut provided are not yet available in the urethra. Future studies should focus on the relationships among nerves, interstitial cells, and the surrounding smooth muscle in the urethra to determine whether the interstitial cells are selectively innervated.
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ACKNOWLEDGEMENTS |
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We thank Action Research and the Wellcome Trust for funding this study.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Hollywood, Smooth Muscle Group, Dept. of Physiology, The Queen's Univ. of Belfast, 97 Lisburn Road, BT9 7BL Belfast, United Kingdom (E-mail: m.hollywood{at}qub.ac.uk; website: www.smoothmusclegroup.org).
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.
May 16, 2002;10.1152/ajpcell.00085.2002
Received 25 February 2002; accepted in final form 3 May 2002.
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