Purinergic activation of spontaneous transient outward
currents in guinea pig taenia colonic myocytes
In Deok
Kong,
Sang Don
Koh, and
Kenton M.
Sanders
Department of Physiology and Cell Biology, University of Nevada
School of Medicine, Reno, Nevada 89557
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ABSTRACT |
Spontaneous transient outward currents
(STOCs) were recorded from smooth muscle cells of the
guinea pig taenia coli using the whole cell patch-clamp technique.
STOCs were resolved at potentials positive to
50 mV. Treating
cells with caffeine (1 mM) caused a burst of outward currents
followed by inhibition of STOCs. Replacing extracellular
Ca2+ with equimolar
Mn2+ caused STOCs to "run
down." Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM)
inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs"
remained in the presence of these drugs. Mini-STOCs were reduced by
apamin (500 nM), an inhibitor of small-conductance Ca2+-activated
K+ channels (SK channels).
Application of ATP or 2-methylthioadenosine 5'-triphosphate
(2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP
persisted in the presence of charybdotoxin but were blocked by
combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not
increase STOCs in the presence of pyridoxal phosphate
6-azophenyl-2',4'-disulfonic acid, a
P2 receptor blocker. Similarly,
pretreatment of cells with U-73122 (1 µM), an inhibitor of
phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin
C, an inositol 1,4,5-trisphosphate
(IP3) receptor blocker,
attenuated STOCs, but these events were not affected by ryanodine. The
data suggest that purinergic activation through P2Y receptors results in localized
Ca2+ release via PLC- and
IP3-dependent mechanisms. Release
of Ca2+ is coupled to STOCs, which
are composed of currents mediated by large-conductance
Ca2+-activated
K+ channels and SK channels. The
latter are thought to mediate hyperpolarization and relaxation
responses of gastrointestinal muscles to inhibitory purinergic stimulation.
calcium sparks; small-conductance calcium-activated potassium
channels; purinergic neurotransmission; P2Y receptors; inositol
1,4,5-trisphosphate receptors
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INTRODUCTION |
MANY TYPES OF CELLS UNDERGO spontaneous, localized
Ca2+ release
(Ca2+ "sparks" or
"puffs"), and local Ca2+
concentration can reach levels sufficient to regulate
Ca2+-dependent conductances in the
plasma membrane (13, 20). In the original communication
describing Ca2+ sparks in smooth
muscles, these events were reported to be coupled to spontaneous
transient outward currents (STOCs; see Ref. 20), which are due to
activation of large-conductance
Ca2+-activated
K+ (BK) channels (4). Recent
experiments have shown that localized Ca2+ transients regulate the open
probabilities of other
Ca2+-dependent conductances, such
as Ca2+-activated
Cl
channels (31). In most
studies of Ca2+ sparks in smooth
muscles, localized Ca2+ release
has been attributed to ryanodine receptors because treatment of cells
with ryanodine blocked spontaneous and agonist-enhanced sparks (16, 20,
24, 31). Recently, however, release of Ca2+ from inositol
1,4,5-trisphosphate (IP3)
receptor-operated stores has also been shown to be a source of
Ca2+ spark activity in smooth
muscles, and there may be a regenerative relationship between
Ca2+ release from
IP3 receptors and ryanodine
receptors (3, 6).
Normally, enhanced production of
IP3 and subsequent
Ca2+ release is characteristic of
responses to excitatory agonists in smooth muscles. However, localized
Ca2+ transients mediated by
IP3 receptors may provide a novel
means by which Ca2+-dependent
ionic conductances are activated by inhibitory agonists. In
gastrointestinal (GI) smooth muscles, this mechanism might explain the
inhibitory actions of ATP, which is thought to be an inhibitory
neurotransmitter released from enteric motoneurons (11, 17).
Stimulation of GI muscle cells with ATP or the
P2Y receptor agonist
2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) leads to
activation of small-conductance
Ca2+-activated
K+ (SK) channels and
hyperpolarization (18, 28). The inhibitory response to ATP in GI
muscles appears to involve occupation of P2Y receptors (9, 29), activation
of phospholipase C (PLC), and enhanced production of
IP3 (2, 8, 21). It is possible that localized Ca2+ release could
provide the link between P2Y
receptors and activation of SK channels.
We previously showed that Ca2+
sparks in murine colonic muscles are elicited by
IP3-dependent mechanisms (3), but
STOCs, the standard electrophysiological assay of
Ca2+ sparks, are due to activation
of BK channels (see Ref. 20 and review in Ref. 7). BK channels do not
mediate the inhibitory responses attributable to ATP in GI muscles,
because inhibitors of BK do not block postjunctional inhibitory
junction potentials (29). Purinergic inhibitory responses appear to be
mainly due to activation of SK channels (18, 28). In the present
study, we attempt to relate the P2Y
receptor-IP3 pathway to activation of SK channels by
investigating whether apamin-sensitive STOCs [not blocked
by charybdotoxin (ChTX) or iberiotoxin] are present in GI muscles
and regulated by purinergic agonists. We use myocytes isolated from the
guinea pig taenia coli for these studies, because this is the classic
preparation in which apamin-sensitive, enteric inhibitory responses
attributed to ATP were first observed (1, 10, 27).
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MATERIALS AND METHODS |
Preparation of smooth muscle cells.
Guinea pigs of either sex were killed by
CO2 asphyxiation. Isolated strips
(1-2 cm long) of taenia coli were incubated in Ca2+-free Hanks' solution (see
Solutions), containing 0.12%
(wt/vol) collagenase (Worthington Biochemical, Freehold, NJ), 0.2%
soybean trypsin inhibitor (type II-S, Sigma, St. Louis, MO), and 0.2% BSA (Sigma). After incubation at 37°C for 15 min with gentle
stirring, tissues were washed four times with enzyme-free Hanks'
solution. The tissue pieces were then triturated with a wide-bore
fire-polished Pasteur pipette to create a cell suspension. The cells
were stored at 4°C and used within 6 h.
Current and voltage measurements.
Drops of the cell suspensions were placed on a glass coverslip forming
the bottom of a 300-µl chamber mounted on an inverted microscope.
Cells were allowed to adhere to the coverslip, and then the chamber was
perfused at a rate of 3 ml/min. "Giga-seals" were made with
fire-polished glass pipettes having tip resistances of 3-4 M
.
The whole cell, perforated patch (amphotericin B) configuration of the
patch-clamp technique was used to record ionic currents under voltage
clamp. An Axopatch 200B amplifier with a CV-4 headstage (Axon
Instruments, Foster City, CA) was used to measure ionic currents and
membrane potentials. Current-clamp experiments (0 current) also were
performed on cells with amphotericin-perforated patches. The changes of
membrane potential were recorded on a chart recorder (Gould 2200S). A
personal computer running pCLAMP software (version 6.0.4, Axon
Instruments) was used to collect data.
Frequencies and amplitudes of STOCs were analyzed using the Mini
analysis program (Synaptosoft Software, Leonia, NJ) for 100 s at given
holding potentials. Amplitude histograms were constructed using a bin
size of 1 pA. All recordings were performed at room temperature
(22-25°C).
Solutions and reagents.
CaCl2, KCl,
KH2PO4,
NaCl, NaHCO3,
Na2HPO4,
sucrose, and glucose were from Fisher Scientific (Fair Lawn, NJ).
(±)Bay K 8644, xestospongin C, U-73122, and U-73343 were purchased
from Calbiochem and dissolved in DMSO. After dilutions, the final
concentration of DMSO was <0.01%. ATP and 2-MeS-ATP were purchased
from Sigma. After the nucleotides were dissolved into HEPES-based
buffer, the pH was corrected to 7.4. All other chemicals were also
purchased from Sigma. Krebs solution contained (in mM) 125 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 15.5 NaHCO3, 1.2 Na2HPO4,
and 11.5 glucose, with pH adjusted to 7.4 by bubbling with 95%
O2 and 5%
CO2.
Ca2+-free Hanks' solution
contained (in mM) 125 NaCl, 5.36 KCl, 15.5 NaHCO3, 0.336 Na2HPO4,
0.44 KH2PO4,
10 glucose, 2.9 sucrose, and 11 HEPES, with pH adjusted to 7.4 with
NaOH. The bath solution (CaPSS) contained (in mM) 135 NaCl, 5.0 KCl,
2.0 CaCl2, 1.2 MgCl2, 10.0 glucose, and 10 HEPES,
with pH adjusted to 7.4 with Tris. The pipette solutions contained (in
mM) 110 potassium gluconate, 30 KCl, 5 MgCl2, and 5 HEPES, with pH
adjusted to 7.2 with Tris.
Statistical analyses.
Data are expressed as means ± SE of
n cells. All statistical analyses were
performed using SigmaStat 2.0 software (Jandel, San Rafael, CA). We
used paired t-tests and one-way
repeated measures ANOVA to compare groups of data. In all statistical
analyses, P < 0.05 was considered
statistically significant.
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RESULTS |
STOCs in isolated myocytes.
Single myocytes from the guinea pig taenia coli were studied with the
amphotericin-perforated patch technique. In preliminary studies, we
noted that STOCs could be evoked when cells were held at depolarized
potentials. STOCs were not resolved at potentials negative to
60
mV. Near
50 mV, small-amplitude STOCs (<10 pA) were recorded.
When cells were held at more positive potentials, STOCs of highly
variable amplitude and frequency were recorded (Fig.
1A).
Amplitude histograms were constructed, and the peak currents of each
distribution (1-pA bin size) were plotted as a function of membrane
potential (Fig. 1, B and
C). The amplitude distributions were
bimodal: the mean amplitude of the large-amplitude STOCs was 19 ± 3 pA, and the mean amplitude of small-amplitude STOCs was 5 ± 1 pA,
at a holding potential of
30 mV
(n = 8). We refer to STOCs of <10 pA
as "mini-STOCs" and STOCs of >10 pA as "large STOCs" at
holding potentials of
40 mV or
30 mV.

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Fig. 1.
Effects of charybdotoxin (ChTX) on spontaneous transient outward
currents (STOCs) in cells studied with amphotericin-permeabilized patch
technique. A: control. Representative
STOCs recorded at test potentials ranging from 50 to 20
mV. At holding potentials of 30 mV or 40 mV, mini-STOCs
were defined as events <10 pA and large STOCs (denoted by *) as
events >10 pA. Inset: expanded scale
showing STOCs (arrows). At potentials more negative than 40 mV,
it was not possible to distinguish between mini-STOCs and large STOCs,
so all events are grouped as mini-STOCs.
B: current-voltage
(I-V)
relationship summarizing STOCs from 8 cells.
C: representative amplitude histogram
of mini- and large STOCs recorded at a holding potential of 30
mV. Amplitude distribution shows a bimodal distribution.
D: ChTX (200 nM; traces were recorded
after 10-min incubation) abolished activity of large STOCs. Mini-STOCs
were still apparent after ChTX (denoted by +).
Inset: remaining mini-STOC in presence
of ChTX. E:
I-V
relationship after treatment with ChTX
(n = 8).
F: representative amplitude histogram
(holding potential 30 mV) shows inhibition of large STOCs and
reduction in mini-STOCs after ChTX treatment.
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STOCs have been reported to result from openings of BK channels, and
these events are initiated by focal release of
Ca2+
(Ca2+ sparks) from intracellular
stores (4, 20). STOCs of varying amplitudes could be due to multiple
Ca2+ spark sites where different
numbers of BK channels are affected or due to multiple populations of
Ca2+-dependent conductances. We
tested the latter hypothesis with channel antagonists to selectively
block specific conductances. Addition of ChTX (200 nM;
n = 5), iberiotoxin (200 nM;
n = 3), or TEA (1 mM;
n = 2), potent inhibitors of BK
channels, completely blocked large-amplitude STOCs (Fig. 1,
D-F).
ChTX also attenuated mini-STOCs from 338 ± 59 to 60 ± 8 events/100 s (82% inhibition) at a holding potential of
30 mV
(Figs. 1 and 2). These data
suggest that mini-STOCs may be partially due to BK channels, but a
ChTX-resistant component also contributed to mini-STOCs. ChTX-resistant
mini-STOCs were completely abolished within 10 min when extracellular
Ca2+ was replaced with equimolar
Mn2+ (Fig.
2C), and STOC activity recovered
when extracellular Ca2+ was
restored (data not shown).

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Fig. 2.
Effects of extracellular Ca2+ on
STOCs. Same cell was held at 30 mV.
A: STOCs recorded under control
conditions (i.e., 2 mM external
Ca2+ concentration).
B: mini-STOCs remaining in presence of
200 nM ChTX for 10 min. C: inhibition
of all STOCs after replacement of external
Ca2+ in
Ca2+-containing physiological
saline solution (CaPSS) with Mn2+
(MnPSS). D: summary of results from 5 cells expressing number of events during 100-s recordings. Data are
grouped in 10-pA bins. * P < 0.05.
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Bay K 8644 (2 µM) markedly increased both types of STOCs
(Fig. 3,
A and
B), but nicardipine (1 µM) did not
affect the occurrence of STOCs (data not shown). Exposing cells to
caffeine (1 mM) caused large "burstlike" episodes of STOCs. The
enhanced STOC activity was followed by a quiescent period (holding
potential
40 mV; Fig. 3, C and
D). Ryanodine (10 µM) did not
significantly affect the occurrence of either type of STOC (holding
potential
30 mV; n = 4; Fig.
4,
A-C).
In contrast, xestospongin C (1 µM), a membrane-permeant blocker of
IP3 receptors (14), significantly
inhibited both large STOCs and mini-STOCs
(n = 4; Fig. 4,
D-F).
These data suggest that STOCs are not caused by, but can be enhanced
by, Ca2+ entry via L-type
Ca2+ channels. The trigger for
STOCs in these cells appears to be release of
Ca2+ from an
IP3 receptor-operated store.

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Fig. 3.
Effects of Bay K 8644 and caffeine on STOCs. Cells were held at holding
potential of 40 mV. A: control.
STOCs recorded in normal CaPSS. B:
after application of Bay K 8644 (2 µM).
C: control. STOCs recorded from a
different cell in normal CaPSS. D:
after application of caffeine (1 mM). Parallel lines denote 2 min or
recording omitted between 5th and 6th traces.
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Fig. 4.
Effects of ryanodine and xestospongin C on STOCs. Cells were held at
30 mV. A: control. STOCs
recorded in normal CaPSS. B: after
addition of ryanodine (10 µM) for 20 min.
C: summary of results showing number
of events during 100-s recordings from 4 cells.
D: control. STOCs in a different cell
in CaPSS. E: after addition of
xestospongin C (1 µM) for 20 min. F:
summary of results showing number of events during 100-s recordings
from 4 cells. * P < 0.05 vs.
control.
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Apamin (500 nM), an antagonist of SK channels (5, 12, 18), attenuated
mini-STOCs (Fig. 5). Mini-STOCs recorded
during 100 s were reduced from 373 ± 32 to 306 ± 23 events
(17.4% inhibition, n = 5, P < 0.05). Large STOCs were also
slightly decreased by apamin treatment; however, this effect did not
reach a level of statistical significance (Fig. 5,
B and
C). It is possible that large-amplitude STOCs contain a component of current contributed by
apamin-sensitive channels. Inhibition of these channels may cause
reduction in the amplitudes of large STOCs and cause these events to be
tabulated as mini-STOCs after apamin. These experiments were conducted
at a holding potential of
30 mV to exclude the potential
involvement of Ca2+-activated
Cl
currents (expected
reversal potential
40 to
30
mV). The data suggest that STOCs in colonic myocytes result from
transient openings of BK and apamin-sensitive SK channels.

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Fig. 5.
Effects of apamin on STOCs. Cell was held at 30 mV.
A: control. STOCs in CaPSS.
B: after addition of apamin (500 nM)
for 10 min. C: summary of results
showing number of events during 100-s recordings from 5 cells.
* P < 0.05 vs. control.
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Activation of STOCs by ATP and 2-MeS-ATP.
Purinergic stimulation by ATP and 2-MeS-ATP activates SK channels in
visceral smooth muscle (18, 28). We tested the effects of these
agonists at a holding potential of
40 mV. ATP (1 mM; n = 4) and 2-MeS-ATP (100 µM;
n = 5) significantly increased the activity of
mini-STOCs (control vs. ATP, from 204 ± 25 to 273 ± 25 events/100 s, a 35% increase; control vs. 2-MeS-ATP, from 174 ± 27 to 257 ± 39 events/100 s, a 50% increase; Fig.
6). Large STOCs also tended to be increased
by ATP (from 10 ± 6 to 23 ± 12 events/100 s) and 2-MeS-ATP
(from 14 ± 8 to 28 ± 14 events/100 s); however, these responses
did not reach levels of statistical significance. We also characterized
purinergic activation of STOCs in the presence of ChTX to block BK
channels. As above, ChTX (200 nM) significantly attenuated large STOCs
and mini-STOCs in these cells (Fig. 7,
A and
B). Addition of 2-MeS-ATP in the
continued presence of ChTX increased the frequency (ChTX vs. ChTX + 2-MeS-ATP, 46 ± 12 vs. 122 ± 15, 222% increase,
n = 4) of mini-STOCs (Fig. 7,
C and
D). Both components of STOCs were
greatly attenuated by joint application of apamin and ChTX (i.e.,
mini-STOCs were reduced from 313 ± 30 to 16 ± 4 events/100 s by
apamin and ChTX, a 95% inhibition; Fig. 8,
A and
B). Large STOCs were completely abolished by apamin and ChTX. In the presence of these drugs, 2-MeS-ATP
did not increase the activity of mini-STOCs or large STOCs (Fig. 8,
C and
D). These data suggest that at least
a portion of the response to purinergic stimulation is due to STOCs
mediated through activation of apamin-sensitive channels.

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Fig. 6.
Effects of ATP and 2-methylthioadenosine 5'-triphosphate
(2-MeS-ATP) on STOCs. Cells were held at 40 mV.
A: control STOCs.
B: after addition of ATP (1 mM).
C: summary of results showing number
of events during 100-s recordings from 4 cells.
D: control. STOCs from a different
cell. E: after addition of 2-MeS-ATP
(100 µM). F: summary of results
showing number of events during 100-s recordings from 5 cells.
* P < 0.05 vs. control.
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Fig. 7.
Effects of 2-MeS-ATP on STOCs in presence of ChTX. Cell was held at
30 mV. A: control. STOCs in
normal CaPSS. B: after addition of
ChTX (200 nM) for 10 min. C: 2-MeS-ATP
(100 µM) increased number of mini-STOCs in presence of ChTX.
D: summary of results showing number
of events during 100-s recordings from 4 cells.
* P < 0.05.
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Fig. 8.
Effects of 2-MeS-ATP on STOCs in presence of ChTX and apamin. Cell was
held at 30 mV. A: control.
STOCs in normal CaPSS. B: after
addition of ChTX (200 nM) and apamin (500 nM) for 10 min.
C: in presence of ChTX and apamin,
2-MeS-ATP (100 µM) failed to increase STOCs.
D: summary of results showing number
of events during 100-s recordings from 4 cells.
* P < 0.05.
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Receptors and second messengers mediating the effect of 2-MeS-ATP on
STOCs.
We tested whether the enhancements in STOCs by ATP and 2-MeS-ATP were
due to activation of purinergic receptors. Pyridoxal phosphate
6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS; 5 µM), an antagonist of P2
receptors (19), decreased large STOCs and mini-STOCs at a holding
potential of
30 mV (Fig. 9,
A and B). 2-MeS-ATP failed to increase the
activity of STOCs in the presence of PPADS (Fig. 9,
C and
D; n = 4).

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Fig. 9.
Effects of 2-MeS-ATP on STOCs in presence of pyridoxal phosphate
6-azophenyl-2',4'-disulfonic acid (PPADS). Cell was held at
30 mV. A: control. STOCs in
normal CaPSS. B: after addition of
PPADS (5 µM, 10-min incubation). C:
in presence of PPADS, 2-MeS-ATP (100 µM) failed to increase STOCs.
D: summary of results showing number
of events during 100-s recordings from 4 cells.
* P < 0.05.
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Cells were pretreated with U-73122 (1 µM), an inhibitor of PLC (see
Ref. 25), for 10 min to determine whether the activation of PLC
mediates the increase in STOCs caused by 2-MeS-ATP. U-73122 decreased
the activity of mini-STOCs (from 304 ± 27 to 224 ± 24 events/100 s, 26% inhibition, n = 4;
Fig. 10). 2-MeS-ATP (100 µM) failed to
increase the activity of STOCs in the presence of this compound (Fig.
10, C and
D; n = 4). U-73343 (1 µM), an inactive form of U-73122, had no effect on
STOCs, and 2-MeS-ATP increased mini-STOCs in the presence of U-73343
(holding potential
30 mV; Fig. 11;
n = 4). 2-MeS-ATP failed to enhance
STOC frequency in cells pretreated with xestospongin C
(n = 2).

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Fig. 10.
Effects of 2-MeS-ATP on STOCs in presence of U-73122. Cell was held at
30 mV. A: control. STOCs in
normal CaPSS. B: after addition of
U-73122 (1 µM, 10-min incubation).
C: 2-MeS-ATP (100 µM) failed to
increase STOCs in presence of U-73122.
D: summary of results showing number
of events during 100-s recordings from 4 cells.
* P < 0.05.
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Fig. 11.
Effects of 2-MeS-ATP on STOCs in presence of U-73343. Cell was held at
30 mV. A: control. STOCs in
normal CaPSS. B: after addition of
U-73343 (1 µM, 10-min incubation).
C: 2-MeS-ATP (100 µM) increased
mini-STOCs in presence of U-73343. D:
summary of results showing number of events during 100-s recordings
from 4 cells. * P < 0.05.
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Hyperpolarization induced by purinergic activation.
In the current-clamp mode (0 current), we investigated the effects of
2-MeS-ATP on membrane potential. At rest, membrane potential averaged
44.2 ± 3.9 mV (n = 7). In three of
seven cells, small fluctuations in resting membrane potential were
observed during resting conditions. Application of 2-MeS-ATP (100 µM)
to these cells induced transient hyperpolarizations (10 ± 3 mV in
amplitude, n = 3; Fig.
12A).
There was variability in the durations of the transient
hyperpolarizations, which may have reflected differences in the degree
of summation of STOCs. In four cells, spontaneous spikelike
hyperpolarizations were observed during resting conditions (e.g.,
frequency averaged 353 ± 29 per 100 s). Addition of ChTX (200 nM)
under current-clamp conditions induced a 3 ± 1 mV depolarization (P < 0.05) and decreased the amplitude (to 5 ± 2 mV, P < 0.05) and frequency
(to 220 ± 23 per 100 s, P < 0.05; see Fig. 12B) of the transient
hyperpolarizations. In the presence of ChTX, 2-MeS-ATP restored the
amplitude of the spontaneous transient hyperpolarizations (to 9 ± 2 mV, P < 0.05 compared with amplitude
in the presence of ChTX) and increased the frequency (to 580 ± 46, P < 0.05; see Fig.
12B). These events were consistent
with the changes in STOC frequency induced by ChTX and 2-MeS-ATP under
voltage-clamp conditions. The data suggest that the hyperpolarizations
caused by 2-MeS-ATP are mediated, in part, by ChTX-insensitive STOCs.

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Fig. 12.
Effects of 2-MeS-ATP on resting membrane potentials of current-clamped
cells (0 current). A: continuous trace
from a representative cell showing transient hyperpolarization after
application of 2-MeS-ATP (100 µM; exposure denoted by bar).
B: discontinuous recording (10 min
were removed between 1st and 2nd traces, as denoted by parallel lines
at end of 1st trace) demonstrating effects of 2-MeS-ATP after prior
addition of ChTX (200 nM). ChTX (black bar over 1st and 2nd traces)
reduced amplitude and frequency of transient hyperpolarizations that
occurred spontaneously in this cell and produced a small depolarization
in membrane potential. In presence of ChTX, 2-MeS-ATP (shaded bar in
2nd trace) increased frequency of transient hyperpolarizations and
restored membrane potential. Dotted line denotes average resting
potential during control phase of recording.
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DISCUSSION |
STOCs have been used as an assay of localized
Ca2+ release (4, 20), and
quantitative analysis has shown a high degree of correlation between
Ca2+ spark amplitude and STOC
amplitude (22). In some smooth muscle cells, however,
Ca2+ sparks are coupled to more
than a single Ca2+-dependent
conductance (31), and the present study indicates that this is also the
case in guinea pig taenia coli smooth muscle cells. Records of STOCs
from taenia coli myocytes showed typical large-amplitude STOCs and
frequent low-amplitude (<10 pA) STOC-like events. All STOCs were
Ca2+ dependent; they were enhanced
by Bay K 8644 and disappeared when cells were incubated for extended
periods in low external Ca2+ or
after intracellular stores were depleted by addition of caffeine. The
data indicated that Ca2+ release
from IP3-sensitive stores may be
the trigger for STOCs in taenia coli myocytes. When BK channels were
blocked by ChTX or iberiotoxin (by addition of >15 times the
dissociation constant for smooth muscle BK channels; see Refs. 15, 23),
STOC-like activity persisted, but the remaining events were of much
smaller amplitude than the events attributable to BK. Mini-STOCs were partially blocked by apamin, suggesting that at least a portion of the
current was due to SK channels. These data suggest that at least two
Ca2+-dependent conductances are
activated by localized Ca2+
release in myocytes from the taenia coli.
The mini-STOCs were relatively difficult to resolve in relation to
STOCs caused by BK channels. This is likely due to the fact that the
conductance of SK channels is comparatively low (i.e., 5.3 pS, see Ref.
18). Thus >40 SK channels would need to open at the same time to
produce the conductance equivalent to 1 BK channel. Because STOCs are
typically attributed to the nearly simultaneous increase in open
probability of many BK channels, a very large number of SK channels
would need to be clustered near sites of
Ca2+ sparks to produce STOCs of an
amplitude equivalent to BK STOCs. Thus it is logical that STOCs due to
SK channels are of much smaller amplitude and typically obscured by the
much larger amplitude BK STOCs. Typically, openings of SK channels
might enhance the amplitude of BK STOCs or produce what would appear to
be increased baseline noise in current records. The need to use
depolarized potentials to resolve the small currents associated with
mini-STOCs increases contamination from BK channels. Therefore,
resolution of STOCs due to SK channels required blocking of BK channels.
It was not possible to separate STOCs due to SK and BK channels purely
on the basis of amplitude. A significant portion (~80%) of the
mini-STOCs were inhibited by ChTX, suggesting that smaller clusters of
BK channels or clusters at some distance from the primary spark sites
may contribute to the low-amplitude population of STOCs. Apamin
inhibited the small-amplitude STOCs by ~20%. Therefore, the
mini-STOCs appear to represent outward currents due to openings of both
BK and apamin-sensitive SK channels.
The mechanisms by which ATP increases cell membrane conductance and
causes hyperpolarization of GI smooth muscles have been unclear.
Previous studies showed that ATP, UTP, and the
P2Y agonist 2-MeS-ATP activated an
outward current under whole cell recording conditions, and this was
attributed to an increase in the open probability of SK channels (18,
28). In the present study, we show that 2-MeS-ATP increases the
frequency of STOCs, and the data suggest that this occurs via
occupation of P2Y receptors (PPADS), activation of PLC (U-73122), and increased
IP3 production (xestospongin C).
The increase in STOCs in response to 2-MeS-ATP suggests that
IP3-dependent amplification of
Ca2+ release (either an increase
in the number of sparking sites or in the frequency of sparking from
established sites) is the main mechanism responsible for
P2Y receptor-mediated stimulation
of outward current and hyperpolarization. Experiments performed in current clamp clearly demonstrate the link between purinergic stimulation and hyperpolarization. In isolated cells, hyperpolarization transients were quantal in nature and most likely the result of the
STOCs recorded under voltage clamp. In intact tissues, the quantal
hyperpolarization transients occurring in many coupled cells would tend
to summate temporally and spatially, yielding continuous, analog
hyperpolarization responses.
We found that PPADS (P2 receptor
blocker) and U-73122 (PLC blocker) also reduce the frequency of
spontaneous STOCs. These data could be interpreted as nonspecific
effects of these compounds on SK channels. Previous studies argue
against the idea that PPADS blocks SK channels. We found that PPADS
decreased the open probability of SK channels in murine colon, but
these channels could be activated by releasing internal stores of
Ca2+ with caffeine (18). PPADS has
been shown to be a partial antagonist of
IP3 receptors (26), and this may
explain why this compound reduced spontaneous STOC activity. The
nonspecific effects of U-73122 were controlled for by also testing
U-73343, a nonactive, structurally similar analog of U-73122. The
inactive analog had no effect on STOC occurrence. It is likely that
U-73122 reduced basal production of
IP3 in the cells, and this might
explain the reduction in STOC frequency in response to this compound.
In studies of mouse colonic myocytes, we showed that spontaneous
Ca2+ sparks are caused by release
of Ca2+ from
IP3 receptor-operated stores (3).
Purinergic stimulation increased
Ca2+ sparks and tended to organize
local release events into Ca2+
waves. Ca2+ sparks and
Ca2+ waves never elevated global
Ca2+ to the threshold for
contraction. A similar enhancement in
Ca2+ spark frequency in response
to 2-MeS-ATP is also likely in taenia coli, because we noted an
increase in STOCs when cells were exposed to this compound.
Ca2+ release is associated with
activation of SK and BK channels in myocytes from the taenia coli. This
raises the question of why tissue responses to purinergic stimuli or
enteric inhibitory neural inputs are reduced by apamin but unaffected
by inhibition of BK channels (30). As was recently pointed out by ZhuGe
and co-workers (31), the ionic conductances activated by
Ca2+ sparks are strongly affected
by the voltage range in which cells are held or are operating during
physiological conditions. These authors found that BK STOCs were
favored at more depolarized potentials, but
Ca2+-activated
Cl
currents (spontaneous
transient inward currents) were more frequently observed at more
negative potentials. These results are partly explained by the driving
forces responsible for outward K+
currents and inward
Cl
currents. However, BK
channels are both voltage and Ca2+
dependent, and the open probability of these channels is very low at
the relatively negative resting membrane potentials of GI muscles
(i.e., negative to
50 mV). SK channels are not voltage dependent, and open probability is increased by low concentrations of
Ca2+ (18). Therefore, the
weighting of purinergic enteric inhibitory responses toward SK channels
may be partially due to the voltage range of resting membrane
potentials of GI muscles. Stimulating cells with 2-MeS-ATP yielded
increases mainly in mini-STOCs, suggesting that
Ca2+ release via
IP3 receptor-operated stores
occurs in close proximity to clusters of SK channels. Therefore, it is
possible that part of the discrimination between activation of SK and
BK channels is due to tight coupling between receptors, second
messenger pathways, IP3
receptor-operated stores, and SK channels. It would be extremely interesting to know whether the clustering of cellular effector proteins are postjunctional specializations associated with sites of
neural innervation in intact muscles.
In summary, the present study suggests a mechanism by which purinergic
neurotransmission causes hyperpolarization and reduces the excitability
of GI smooth muscles. The data are consistent with the following.
Occupation of P2Y receptors
activates PLC and increases production of
IP3. Rather than generally
increasing global cytoplasmic
Ca2+,
IP3 generated via this mechanism
enhances localized Ca2+
transients. This leads to enhanced production of STOCs, which are
mainly caused by activation of SK channels. STOCs cause
hyperpolarization transients that summate and cause the
hyperpolarization responses in intact muscles. Hyperpolarization of GI
muscles lowers excitability and decreases the net open probability of
L-type Ca2+ channels, leading to a
net inhibitory effect.
 |
ACKNOWLEDGEMENTS |
This research was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Program Project Grant PO1-DK-41315.
 |
FOOTNOTES |
Address for reprints and other correspondence: K. M. Sanders, Dept. of
Physiology and Cell Biology, University of Nevada School of Medicine,
Reno, NV 89557 (E-mail: kent{at}physio.unr.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 4 June 1999; accepted in final form 24 September 1999.
 |
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