Small-conductance
Ca2+-dependent
K+ channels activated by ATP
in murine colonic smooth muscle
S. D.
Koh,
G. M.
Dick, and
K. M.
Sanders
Department of Physiology and Cell Biology, University of Nevada
School of Medicine, Reno, Nevada 89557
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ABSTRACT |
The patch-clamp
technique was used to determine the ionic conductances activated by ATP
in murine colonic smooth muscle cells. Extracellular ATP, UTP, and
2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increased
outward currents in cells with amphotericin B-perforated patches. ATP
(0.5-1 mM) did not affect whole cell currents of cells dialyzed
with solutions containing ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid. Apamin (3 × 10
7
M) reduced the outward current activated by ATP by 32 ± 5%. Single channel recordings from cell-attached patches showed that ATP, UTP, and
2-MeS-ATP increased the open probability of small-conductance, Ca2+-dependent
K+ channels with a slope
conductance of 5.3 ± 0.02 pS. Caffeine (500 µM) enhanced the open
probability of the small-conductance K+ channels, and ATP had no effect
after caffeine. Pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS,
10
4 M), a nonselective
P2 receptor antagonist, prevented
the increase in open probability caused by ATP and 2-MeS-ATP. PPADS had
no effect on the response to caffeine. ATP-induced hyperpolarization in
the murine colon may be mediated by
P2y-induced release of Ca2+ from intracellular stores and
activation of the 5.3-pS
Ca2+-activated
K+ channels.
ion channels; apamin; caffeine; purinergic receptors; enteric
inhibitory neurotransmission; gastrointestinal motility
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INTRODUCTION |
MANY VISCERAL TISSUES are innervated by motoneurons
that do not utilize adrenergic or cholinergic transmitters
(nonadrenergic noncholinergic neurotransmission) (for reviews see Refs.
20 and 35). Recent evidence suggests that nitric oxide (NO) is a major
neurotransmitter mediating nonadrenergic noncholinergic inhibition in
many autonomically innervated tissues (9, 28, 34); however, other
transmitters, such as ATP, vasoactive intestinal polypeptide (VIP), and
pituitary adenylyl cyclase-activating peptide (PACAP), are coexpressed
in inhibitory neurons and may be coreleased (10, 18, 29, 31). Many
studies of gastrointestinal (GI) muscles have described at least two
components of hyperpolarization [i.e., inhibitory junction
potentials (IJPs)] in postjunctional cells in response to
inhibitory nerve stimulation (19, 23, 36, 38). Typically, IJPs consist
of an initial fast component of hyperpolarization that is usually
attributed to the release of ATP. The fast component of the IJP is
variably sensitive to apamin (20). In some preparations the fast
component is totally blocked by 100 nM apamin, but in other
preparations the fast component is far less sensitive (23).
Superimposed on, and extending beyond, the fast component is a slower
component that is attributed to release of NO and, possibly, VIP (19,
23, 38). Different species and tissues manifest different degrees of
the slow and fast components, suggesting considerable heterogeneity in
the postjunctional receptors and mechanisms linked to neurally evoked inhibition. For example, in the canine colon, enteric inhibitory inputs
are mediated primarily by NO (13, 39) via activation of a variety of
K+ channels (24), and
apamin-sensitive responses are not present (37). In mouse and human GI
muscles, apamin-sensitive and -insensitive components are present (23,
36). Although there has been speculation that the apamin-sensitive
responses are mediated by ATP, the conductance(s) responsible for the
fast component of IJPs has not been described. Therefore, it is unclear
how ATP causes smooth muscle hyperpolarization.
It is thought that IJPs are due to the activation of
K+ channels (40), and many authors
have speculated that ATP activates small-conductance
Ca2+-dependent
K+ (SK) channels similar to those
expressed in skeletal muscles (7), brain (25), and hepatocytes (11),
because IJPs and responses to ATP are reduced by apamin (1), an
inhibitor of SK channels. Apamin-sensitive and -insensitive isoforms of
SK channels have been identified (25), and therefore it is possible that the apamin-insensitive portion of ATP responses and fast IJPs
could be mediated via apamin-insensitive SK channels. The molecular
structure of SK channels has recently been determined, and functional
channels have been cloned from mammalian brain (25). SK channels are
Ca2+ sensitive and voltage
independent and have unitary conductances of 5-20 pS. These are
characteristic properties that could be used to identify SK channels in
GI smooth muscles.
In the present study we examined the effects of ATP on outward currents
of murine colonic muscle cells to identify the conductance(s) that may
mediate ATP-dependent inhibitory responses in the GI tract. We have
used the patch-clamp technique and compared responses in dialyzed and
"perforated-patch" cells to test the involvement of intracellular
signaling mechanisms. Single channel recordings were also performed to
identify specific K+ channels
activated by ATP.
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METHODS |
Cell preparation.
Colonic smooth muscle cells were prepared from 20- to 30-day-old Balb/C
mice of either sex. Mice were anesthetized with chloroform and killed
by cervical dislocation, and the proximal colon was quickly removed.
Colons were cut open along the longitudinal axis, pinned out in a
Sylgard-lined dish, and washed with
Ca2+-free Hanks'
solution containing (in mM) 125 NaCl, 5.36 KCl, 15.5 NaHCO3, 0.336 Na2HPO4,
0.44 KH2PO4,
10 glucose, 2.9 sucrose, and 11 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES). After removal of the mucosa and submucosa, pieces of
muscle were incubated in a
Ca2+-free Hanks' solution
containing 4 mg/ml fatty acid-free bovine serum albumin (Sigma
Chemical), 14 U/ml papain (Sigma Chemical), 230 U/ml collagenase
(Worthington), and 1 mM dithiothreitol (Sigma Chemical). Tissues were
incubated at 37°C in enzyme solution for 8-12 min and then
washed with Ca2+-free Hanks'
solution. Tissue pieces were gently triturated to create a cell
suspension. Dispersed cells were stored at 4°C in Ca2+-free Hanks' solution
supplemented with minimum essential medium for suspension culture
(Sigma Chemical) and (in mM) 0.5 CaCl2, 0.5 MgCl2, 4.17 NaHCO3, and 10 HEPES. The cells
were allowed to adhere to the bottom of a recording chamber on an
inverted microscope for 5 min before commencement of experiments. All
experiments were performed within 6 h of dispersion of cells.
Voltage-clamp experiments.
Dialyzed whole cell, perforated-patch whole cell, and single channel
voltage-clamp experiments were performed on murine colonic smooth
muscle cells. Currents were amplified with a List EPC-7 amplifier and
digitized with a 12-bit analog-to-digital converter (model TL-1, DMA
interface, Axon Instruments). Data were stored on videotape or
digitized on-line using pCLAMP software (version 5.5.1 or 6.03, Axon
Instruments). Data were sampled at 1-5 kHz and low-pass filtered
at 0.2-1 kHz using an eight-pole Bessel filter. Probability
density plots were obtained by scaling the amplitude histograms so that
the total area under the curve was equal to 1.0. In the experiments
where the Ca2+ sensitivity of the
small-conductance K+ channels was
studied in excised patches, a sampling rate of 1 kHz and a filtration
rate of 0.2 kHz were used. Variations in the baseline were corrected
with ASCD software (Dr. G. Droogmans, KU, Louvain, Belgium). After
baseline adjustment, all-points amplitude histograms were constructed
and open probability was determined. A sampling period of 2 min was
used for these analyses.
Solutions.
For the recordings of K+ currents
with the dialyzed whole cell technique, the external
Mn2+-containing physiological
saline solution (MnPSS) consisted of (in mM) 5 KCl, 135 NaCl, 2 MnCl2, 10 glucose, 1.2 MgCl2, and 10 HEPES adjusted to pH
7.4 with tris(hydroxymethyl)aminomethane (Tris). In some experiments,
MnCl2 was replaced with
CaCl2
[Ca2+-containing
physiological saline solution (CaPSS)]. Composition of the
internal solution for dialyzed cells was (in mM) 110 potassium gluconate, 20 KCl, 5 MgCl2, 2.7 K2ATP, 0.1 Na2GTP, 2.5 creatine phosphate
disodium, 5 HEPES, and 1 ethylene glycol-bis(
-aminoethyl ether)-N,N, N',N'-tetraacetic
acid (EGTA) adjusted to pH 7.2 with Tris. For perforated whole cell
patch-clamp experiments, composition of the pipette solution was (in
mM) 110 potassium gluconate, 20 KCl, 0.5 EGTA, and 5 HEPES adjusted to
pH 7.2 with Tris. Amphotericin B (90 mg/ml, Sigma Chemical) was
dissolved with dimethyl sulfoxide, sonicated, and diluted in the
pipette solution to give a final concentration of 270 µg/ml. External
solution for these experiments was the same as for the dialyzed whole
cell patch-clamp experiments. In some experiments, 3 × 10
7 M apamin (Sigma
Chemical) was added to the external solution. Freshly prepared
K2ATP, UTP, and
2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) were applied to
cells via addition to the bath solution and superfusion.
For recording K+ channel currents
in cell-attached or excised patches, the bath solution contained (in
mM) 140 KCl, 1 EGTA, 0.61 CaCl2,
and 10 HEPES adjusted to pH 7.4 with Tris. Various concentrations of
Ca2+ were added to bath solutions
buffered by 1 mM EGTA to create Ca2+ activities from
10
5 to
10
9 M. Activities were
calculated with a program developed by C.-M. Hai (University of
Virginia, Charlottesville, VA). In these experiments the pipette
solution was identical to the bath solution, except 200 nM
charybdotoxin was added to the pipette to inhibit large-conductance K+ (BK) channels in most on-cell
experiments. In some experiments, KCl was replaced with NaCl
(equimolar) to study ion selectivity. K+ concentration
([K+]) gradients are
given as external [K+]
divided by internal
[K+]. Caffeine (500 µM; Sigma Chemical) and
10
5-10
4
M pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid
tetrasodium (PPADS; Research Biochemicals) were added to bath solutions
in some experiments.
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RESULTS |
ATP effects in dialyzed whole cell configuration.
The effects of ATP on outward current were first tested by external
application of 1 mM ATP to cells voltage clamped with the conventional,
dialyzed, whole cell configuration of the patch-clamp technique. EGTA
(1 mM) was added to the pipette solution to buffer intracellular
Ca2+. In the presence of external
MnPSS (see METHODS), outward
currents were generated by depolarizing test potentials from a holding potential of
80 mV. The outward currents reached a peak and
inactivated during 150-ms test depolarizations. Data generated from
inactivation protocols were fitted with a Boltzmann equation:
y = 1/{1 + exp[(V
Vh)/k]},
where k represents the slope factor
and Vh is voltage of 50% inactivation, which averaged
44 ± 1 mV
(n = 5, data not shown). The
time-dependent decay of the outward current was fit with a single time
constant (
= 62 ± 2 ms at +20 mV; data not shown). At the end of
150-ms test potentials, the outward current had inactivated to 30% of
the peak current. Application of external ATP had no effect on the
outward currents elicited in dialyzed cells. Similar observations were
made when CaPSS was used as the external solution:
1) inactivating outward currents
were elicited by step depolarizations from
80 mV (Fig.
1, A and
B);
2) the sustained current was 26% of
the peak current at the end of 150-ms test pulses; and
3) ATP did not alter the amplitude
of the peak or the sustained current
(n = 4; Fig. 1,
B-D).

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Fig. 1.
Effect of ATP on outward currents in dialyzed cells.
A: current responses of a dialyzed
cell to voltage-clamp steps from 80 to +80 mV in 10-mV
increments. Depolarization evoked large, inactivating outward currents.
B: addition of 1 mM ATP in presence of
Ca2+-containing physiological salt
solution (PSS) had no effect on outward current.
C: summary of peak current responses
from 4 cells. , Cells under control conditions; , responses after
addition of ATP. Data show lack of effect of ATP on peak outward
current in dialyzed cells. D: summary
of sustained current responses (at end of 150-ms pulses); symbols as in
C. Symbols and error bars from data
sets before and after addition of ATP are essentially superimposed.
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Effects of ATP on cells using the perforated-patch, whole cell
configuration.
In the presence of MnPSS, cells voltage clamped with the
perforated-patch technique (34) showed a pattern of current responses similar to the currents observed in experiments on cells using the
dialyzed, whole cell configuration (i.e., peak and sustained currents).
Application of 1 mM ATP activated outward currents in cells in which
the perforated-patch technique was used (Fig. 2, A and
B). ATP produced a sustained outward
component of current, as shown by difference currents (Fig.
2B). For example, at +40 mV the
outward current was enhanced 18 ± 4% at the end of a 500-ms test
depolarization (n = 6). The difference
currents elicited by ATP were well fit by the Goldman-Hodgkin-Katz
(GHK) equation for K+
(r2 = 0.9726, n = 6; Fig.
2C). ATP induced a
K+ permeability of 8.3 × 10
15
cm · s
1 · cm
2.

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Fig. 2.
ATP (1 mM) and 100 µM 2-methylthioadenosine 5'-triphosphate
(2-MeS-ATP) increase outward currents in amphotericin-perforated cells.
A: current records in response to
depolarization from 80 to 40, 0, and +40 mV. , current
records before ATP; , currents in presence of 1 mM ATP.
B: difference currents (i.e., control
currents subtracted from currents in presence of ATP); outward currents
activated by ATP are described. C:
summary current-voltage (I-V) curve
for difference currents obtained from 6 cells. Data points of
I-V relationship were fit by
Goldman-Hodgkin-Katz (GHK) equation for
K+ gradient. From fit of data, it
was calculated that ATP induced a
K+ permeability of 8.3 × 10 15
cm · s 1 · cm 2.
ATP-dependent current was resolvable at potentials negative to range in
which voltage-dependent currents were activated (see Fig.
1C).
D: currents generated in a cell bathed
with Mn2+-containing PSS using
permeabilized-patch technique. Control responses ( ) and responses of
same cell in presence of 100 µM 2-MeS-ATP ( ) are superimposed.
E: difference currents describing
outward currents activated by 2-MeS-ATP.
F:
I-V relationship for difference
currents in E
(n = 4). Data points are fit with GHK
equation for K+.
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Application of 100 µM 2-MeS-ATP, a
P2y receptor agonist, activated
outward currents with characteristics similar to the currents elicited
by ATP (Fig. 2D). 2-MeS-ATP evoked a
sustained outward current (Fig. 2E).
For example, at +40 mV the outward current was enhanced 24.5 ± 2%
at the end of a 500-ms test depolarization (n = 4). The difference currents
elicited by 2-MeS-ATP were well fit by the GHK equation for
K+
(r2 = 0.9716;
Fig. 2F), suggesting that the
current induced by 2-MeS-ATP was due to a
K+-selective conductance. UTP (1 mM) elicited a current similar to the currents elicited by ATP and
2-MeS-ATP; however, lower concentrations of UTP (i.e., 100 µM) had no
effect (n = 2 each dose; data not
shown). The currents activated by ATP and 2-MeS-ATP were not affected
by 5 mM 4-aminopyridine (4-AP) or the combination of 5 mM 4-AP and 10 mM tetraethylammonium (data not shown).
In the presence of CaPSS, the pattern of the outward currents elicited
by depolarization of cells using the perforated-patch technique
changed. Stepping from
80 to +50 mV in 10-mV increments induced
outward currents that reached a peak early in the test pulse, partially
relaxed, and then increased during the remainder of 500-ms test
depolarizations. Thus the inactivating trend observed when cells were
bathed in MnPSS was masked by the development of an additional, slowly
activating component of current in CaPSS (Fig.
3A). ATP
significantly increased the amplitude of the outward currents elicited
in these cells (e.g., 52 ± 6% at +40 mV,
n = 5; Fig. 3,
B and
C). We also noted that significant
inward tail currents developed on repolarization from test potentials
in the presence of ATP (Fig. 3B),
suggesting that, in CaPSS, inward currents, possibly due to
Cl
or nonselective cation
conductances, were also activated. Under these conditions the
current-voltage (I-V) relationship
of the difference current elicited by ATP was not well fit by the GHK equation for K+, suggesting that
ATP activated more than a single ionic conductance (Fig.
3D).

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Fig. 3.
Effects of ATP on perforated-patch cells in presence of extracellular
Ca2+.
A: control currents generated by
depolarization of an amphotericin-permeabilized cell bathed in
Ca2+-containing PSS.
B: current records in same cell after
addition of 1 mM ATP. C: difference
currents; ATP increased outward current during depolarization steps.
Small tail currents noted on repolarization are suggestive of a
Cl or nonselective cation
conductance that may also have been activated by ATP.
D: summary plot of
I-V relationship of difference
currents obtained from 5 cells. Data were fit with GHK equation for
K+. Data were not fit well by GHK
equation, suggesting that ATP activated more than a single conductance
in Ca2+-containing PSS.
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Effect of apamin on ATP-activated currents.
Apamin (3 × 10
7 M),
an inhibitor of SK channels, was applied to the bath solution to test
the pharmacological sensitivity of the current induced by ATP. Apamin
itself had no effect on the outward current elicited in dialyzed cells
(data not shown). Cells with permeabilized patches were stepped from a
holding potential of
80 to +80 mV in 20-mV steps (Fig.
4). ATP (1 mM) increased outward currents,
and apamin partially attenuated the response to ATP (32 ± 5%,
n = 5; Fig. 4,
A and
B). Under control conditions, peak
outward current averaged 760 ± 2 pA and ATP increased current amplitude to 889 ± 2 pA. In a series of time control experiments the outward current activated by ATP declined very little during 11 min
of exposure (i.e., to 862 ± 4 pA,
n = 5). In another group of five cells
with average outward currents of 756 ± 4 pA, ATP increased the
outward current to 884 ± 4 pA. Apamin decreased the outward current
in these cells to an average of 827 ± 3 pA (n = 5).

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Fig. 4.
Apamin attenuated outward current activated by ATP.
A: currents generated by
depolarization of a cell bathed in
Mn2+-containing PSS using
permeabilized-patch technique. Trace
a: control record; trace
b, increase in outward current produced by ATP;
trace c: reduction in outward current
after addition of 3 × 10 7 M apamin in continued
presence of ATP in another cell. Apamin partially blocked ATP-induced
current. B: peak currents in response
to repetitive steps from 80 to 0 mV. , Time course of average
responses to sustained exposure to ATP; , reduction in current
caused by application of 3 × 10 7 M apamin. Points are
averages ± SE from 5 cells exposed continuously to ATP (for at
least 800 s) and 5 cells in which apamin was added during continuous
exposure to ATP.
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K+ channels
activated by ATP.
Single channel studies were performed on 198 patches to identify
K+ channels activated by ATP.
Application of 1 mM ATP to the external solution increased the open
probability of a small-conductance channel in cell-attached patches (32 of 52 patches tested with this protocol). At a patch potential of
60 mV in the presence of
10
7 M extracellular free
Ca2+, few spontaneous channel
openings were noted. However, brief openings of a small-conductance
channel (mean open time = 2 ± 0.5 ms) were occasionally observed.
Because of the brief open times of these channels, we could not fully
resolve the amplitude of the currents (Fig.
5A). ATP
increased open channel probability of the small-conductance channels.
The amplitude of the unitary conductance was 0.35 pA at
60 mV.
After 3 min of exposure to ATP, open probability was 0.14 ± 0.05 (Fig. 5, B and
F), and, after 7 min, open
probability reached a maximum level of 0.26 ± 0.07 (Fig.
5C). The increase in open
probability resulted from an increase in mean open time (i.e., to
29 ± 3 ms), and multiple channel openings were also
observed. Open probability of the channels decreased when ATP was
removed from the external solution. Within the 1st min after removal of
ATP, open probability decreased to 0.13 ± 0.06 (Fig.
5D). Several minutes of washout were
required to return open probability to the control level (Fig.
5E). Figure 5F shows the changes in open
probability elicited by ATP as a function of time
(n = 7 patches in which the entire
exposure and washout of ATP were accomplished).

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Fig. 5.
ATP activated a small-conductance channel in cell-attached patches.
A-E: representative excerpts of a
continuous on-cell patch-clamp recording before and during exposure to
ATP. A: current under control
conditions, i.e., symmetrical 140 mM
K+ and holding potential of
60 mV. Very brief openings of a small-conductance channel were
observed (downward deflections). B: 1 mM ATP increased open probability, and a peak level was reached in
C. D
and E: effect of ATP on channel
activity was reversible on washout. F:
plot of channel activity, expressed as open channel probability
(NPo), as a
function of time (n = 7); letters
(A-E) indicate times at which
traces in A-E were recorded.
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I-V relationship of ATP-induced current.
To further characterize the small-conductance channels activated by
ATP, we performed steady-state recordings and step protocols on
inside-out excised patches. Ionic conditions included symmetrical (140 mM K+ in pipette and bath
solution) and asymmetrical (5 mM
K+ in pipette and 140 mM
K+ in bath solution)
K+ gradients with
10
7 M extracellular free
Ca2+. With the inner surface of
the patch exposed to this concentration of
Ca2+, spontaneous openings of the
small-conductance channels were observed. Amplitudes of the currents
were plotted as a function of potential (Fig.
6C), and
the slope conductance in symmetrical K+ solutions was determined by
linear regression to be 5.3 ± 0.02 pS
(n = 4). Because of the small
amplitude of the currents, it was difficult to resolve single channel
openings at potentials positive to
40 mV in symmetrical
K+ solutions (Fig.
6A).

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Fig. 6.
I-V relationship of small-conductance
K+ channel in excised membrane
patches. A: steady-state recordings
from an inside-out patch with symmetrical 140 mM
K+ and
Ca2+ buffered to
10 7 M. Solid line and c
indicate 0 current when channels were closed; channel openings are
represented by downward deflections.
B: recordings made in asymmetrical
K+ (5 mM in pipette and 140 mM in
bath solution at inside surface of patch). c, Closed; o, open. Data
were sampled at 1 kHz and filtered at 0.2 kHz.
C:
I-V relationship for currents in
experiments depicted in A ( ) and
B ( ). Data points were fitted with
GHK equation. Note shift in extrapolated reversal potentials that would
be predicted if channels were K+
selective.
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To confirm that the currents were due to activation of a
K+ conductance, step
depolarizations were also performed in asymmetrical K+ solutions (i.e., 5 mM
K+ in pipette and 140 mM
K+ in bath solution). In these
experiments we noted three unitary conductances in excised patches:
1) a large-conductance channel (identified as BK channels by biophysical and pharmacological properties; data not shown), 2) a
13-pS channel that showed inactivation and sensitivity to 4-AP (delayed
rectifier channels; data not shown), and
3) small-conductance channels that
corresponded to the 5.3-pS channels observed in symmetrical
K+ solutions (Fig.
6B). The currents resulting from
these channels were fitted with the GHK equation and had a chord
conductance of 2.9 pS at 0 mV (Fig. 6,
B and
C). The calculated equilibrium potential for Cl
and the
theoretical reversal potential for nonselective cation channels under
these conditions were zero, suggesting that the 5.3-pS channels were
not Cl
or nonselective
cation channels.
Ca2+
sensitivity of the 5-pS
K+ channels.
The Ca2+ sensitivity of the 5.3-pS
K+ channels was assessed by
exposing the intracellular surface of excised membrane patches held at
60 mV to several concentrations of free
Ca2+
(n = 4). When
Ca2+ was increased from
10
9 to
10
8 M, there was no change
in open probability (Fig. 7,
A and
B, traces
a and b). At
10
7 M
Ca2+, very brief channel openings
were observed (Fig. 7, A and
B, trace c), but open probability
was low (Fig. 7C). When
Ca2+ was increased to
10
6 M, two to three
channels were activated and open time increased [53 ms vs. 2 ms
(control); Fig. 7, A,
B, trace d, and
D]. At
10
5 M free
Ca2+ the number of channel
openings increased additionally (Fig. 7, A and B, trace
e). The effects of increased
Ca2+ concentration were fully
reversible, and reducing Ca2+ back
to very low concentrations
(10
8 M) decreased open
probability in a concentration-dependent manner. Figure
7C shows a plot of open probability
vs. Ca2+ concentration, and the
50% effective concentration, calculated from the averaged data, was
4.9 × 10
7 M
Ca2+.

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Fig. 7.
Ca2+ sensitivity of
small-conductance channel in inside-out excised patches. Intracellular
surface of an inside-out membrane patch was exposed to
10 9-10 5
M free Ca2+.
A: excerpts of current records while
free Ca2+ concentration in bath
solution was changed to levels indicated above and below current
traces. Open probability of 5.3-pS channel was increased as free
Ca2+ increased.
B: parts of trace in
A are expanded (as indicated by
a-h in
A), and unitary conductance levels
are estimated by dotted lines (solid line and c indicate closed state).
At 10 6 and
10 5 M
Ca2+, openings of at least 2 channels were evident. C: plot from 4 experiments of open probability vs. logarithm of free
Ca2+ concentration
([Ca2+]); 50%
effective concentration (EC50)
was calculated to be 4.9 × 10 7 M. D: all-points histogram obtained from
records taken in presence of
10 6 M free
Ca2+; opening of at least 2 channels is demonstrated.
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Activation of 5-pS
K+ channels by
caffeine.
The fact that open probability of the 5-pS
K+ channels was increased by
intracellular Ca2+ suggests that
entry of Ca2+ or release of
Ca2+ from intracellular stores
could mediate the activation of these channels by ATP in the
cell-attached configuration. Therefore, we tested the effects of
caffeine to determine whether release of stored
Ca2+ might activate the 5.3-pS
channels. Caffeine (500 µM) was applied in the external bath
solution, and single channel activity was monitored while stepping from
80 to 0 mV in the cell-attached configuration with an
asymmetrical K+ gradient (i.e., 5 mM K+ in the pipette and 140 mM
K+ in the bath solution). Three
K+ channels, with conductances of
250, 13, and 5.3 pS (Fig.
8A), were observed on step depolarizations. The open probability of 13-pS
channels, likely to be delayed rectifier channels (see above), was
unaffected by ATP or caffeine. The 250-pS channels, identified as BK
channels, were activated by caffeine treatment (open probability = 0.01 ± 0.01 and 0.12 ± 0.02 for control and caffeine treatment, respectively, n = 4). The increase in
BK open probability was taken as an indication that the caffeine
treatment released intracellular Ca2+. Caffeine also increased open
probability of the 5.3-pS channels (Fig. 8,
B and
B'), which occurred in a
biphasic manner. After application of caffeine, there was an initial
increase in the openings of the 5.3-pS channels; however, this effect
decreased within 3 min. After exposure to caffeine (8 min), addition of ATP had no effect on 5.3-pS channels (data not shown).

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Fig. 8.
Caffeine activated 5.3-pS and large-conductance
K+ (BK) channels in on-cell
patches. A: control recordings from an
on-cell patch. Patch was stepped from 80 to 0 mV. Three unitary
conductances were noted in this patch. Trace
a: opening of 5.3-pS channel; trace
b: opening of 13-pS channel (o, open state; c, closed
state). Openings of BK channels (large transitions) can also be
observed. B: 500 µM caffeine, which
releases Ca2+ from intracellular
stores, increased open probability of BK channels (confirming that
caffeine increased intracellular
Ca2+) and 5.3-pS channels.
Caffeine did not affect 13-pS channels.
A' and
B': all-points amplitude
histograms before and after caffeine treatment, respectively. In
A', histogram was well fit with
a Gaussian distribution (solid line); histogram in
B' was not well fit by same
function as a result of activation of 5.3-pS channels.
|
|
Effects of 2-MeS-ATP and UTP on 5.3-pS
K+ channels.
To observe the effects of more specific agonists of
P2y receptors, we also tested
whether 2-MeS-ATP and UTP enhanced the open probability of the 5.3-pS
channels. 2-MeS-ATP at 10 µM (n = 3) and 100 µM (n = 4) increased the
open probability of the 5.3-pS channel in cell-attached patches. Figure
9 also shows all-points amplitude
histograms for control conditions and the response to 2-MeS-ATP
stimulation (open probability = 0.55 ± 0.11, n = 4) at
60 mV. UTP (1 mM;
n = 3) also increased the open
probability of the 5.3-pS channels; however, 100 µM UTP was without
effect (n = 3; data not shown).

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[in this window]
[in a new window]
|
Fig. 9.
Activation of 5.3-pS channels by 2-MeS-ATP.
A: representative excerpts from an
on-cell recording held at 60 mV.
B: continuous recording after addition
of 100 µM 2-MeS-ATP. This caused a significant enhancement in open
probability of small-conductance channels (closed state indicated by
solid line). A' and
B': current amplitude histograms
for recordings in A and
B, respectively. Note significant
increase in open probability
(Po) for 5.3-pS
channel.
|
|
Effects of PPADS on activation of 5.3-pS
K+ channels.
Because 2-MeS-ATP increased the open probability of the 5.3-pS channel,
we tested whether PPADS, a P2
receptor blocker (27), inhibited this effect. PPADS
(10
5 or
10
4 M) was added to the
external solution, and single channel activity was recorded in the
cell-attached configuration. PPADS itself did not change the activity
of the 5.3-pS channels under control conditions (Fig.
10, A
and B). In the presence of PPADS,
application of 1 mM ATP or 100 µM 2-MeS-ATP did not increase the open
probability of 5.3-pS K+ channels
(Fig. 10C). Caffeine was able to
activate 5.3-pS channels in the presence of PPADS (open probability = 0.18 ± 0.07, n = 4; Fig.
10D).

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[in a new window]
|
Fig. 10.
Pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid
tetrasodium (PPADS) prevented enhancement in 5.3-pS channel open
probability in response to 2-MeS-ATP.
A: 4 traces under control conditions
from a cell-attached patch held at 60 mV. Note small downward
deflections in records, which indicate brief openings of
small-conductance K+ channels.
B: continuation of record after
addition of 100 µM PPADS. PPADS alone did not alter channel activity.
C: PPADS prevented stimulatory effect
of 100 µM 2-MeS-ATP on 5.3-pS channel.
D: addition of 500 µM caffeine
increased channel activity in presence of PPADS.
Right: all-points amplitude
histograms.
|
|
 |
DISCUSSION |
Stimulation of intrinsic enteric inhibitory nerves leads to
hyperpolarization and relaxation of GI muscles (6, 20). There has been
considerable work to determine the transmitters that mediate enteric
inhibitory responses in these muscles, and ATP, VIP, PACAP, and NO have
been considered candidates. In fact, enteric inhibitory
neurotransmission appears to be accomplished by at least two
transmitters released in parallel from the same population of
inhibitory motoneurons. The postjunctional mechanisms that mediate the
inhibitory effects are not well understood, but this question has
importance, because the postjunctional receptors and effectors (i.e.,
ion channels or other cellular mechanisms) may be excellent targets for
therapeutic agents to regulate GI motility. Recent work has described
several K+ channels in canine
colonic muscles that are activated by NO (24), and the present study
has addressed the conductance activated by purinergic agonists. ATP and
the P2y receptor agonist 2-MeS-ATP activated 5.3-pS Ca2+-activated
K+ channels. These channels had
properties similar to SK channels, the small-conductance
Ca2+-activated
K+ channels recently cloned from
mammalian brain (25). Besides their high sensitivity to
Ca2+, SK channels are voltage and
time independent. These channels are ideal candidates for activation of
outward current at the negative resting potentials of GI muscles. ATP
was found to activate outward current at all potentials positive to the
K+ equilibrium potential (Fig. 2).
A portion of the outward current activated by ATP and 2-MeS-ATP was
blocked by apamin, and apamin is known to reduce the fast component of
IJPs in GI muscles of many species (20). Thus channels with properties
consistent with SK channels are expressed by murine colonic myocytes,
and these channels may mediate the fast component of IJPs in GI
muscles. The data from the present study and the work of others are
consistent with the following hypothesis for ATP-dependent
hyperpolarization: 1) ATP activates
P2y receptors;
2)
P2y receptors lead to mobilization of internal Ca2+;
3) increased intracellular
Ca2+ (most likely within the
submembranous compartment) activates SK channels; and
4) enhanced
K+ conductance brings membrane
potential transiently closer to the K+ equilibrium potential.
It is quite clear that the BK channel, another type of
Ca2+-activated
K+ conductance channel, is not
responsible for ATP-dependent hyperpolarization, because blockers of
this channel do not affect IJPs (S. Ward, personal communication).
Ca2+-activated
K+ channels with conductances
smaller than BK channels have been found in the rabbit portal vein
(22), taenia coli (21), and jejunum, guinea pig mesenteric artery (4),
and canine colon (24), but the channel conductances (63-150 pS)
are much greater than the conductance activated by ATP in murine
colonic smooth muscles (i.e., 5.3 pS). The medium-conductance
Ca2+-activated
K+ channels have been reported to
be insensitive to apamin (8, 47). Apamin blocks some isoforms of SK
channels (25) and reduces responses to inhibitory transmitters and ATP
in a variety of GI muscles, including guinea pig stomach (26, 43),
taenia coli (30, 43, 46), and ileum (48). This suggests that a portion of the responses to enteric inhibitory transmitters may be due to
activation of SK channels. The present study documents the expression
of a small-conductance,
Ca2+-activated
K+ channel with properties
consistent with SK channels in GI muscles.
ATP released from nerves could activate different types of purinergic
receptors expressed by smooth muscle cells. Others have documented
excitatory effects of ATP released from extrinsic sympathetic neurons
in colonic muscle, and these effects are mediated by
P2x receptors (41).
P2x receptors are ligand-gated
nonselective cation channels (5, 16, 44). Therefore, it is unlikely that P2x receptors could mediate
IJP responses, because activation of nonselective cation channels would
result in inward current and depolarization (14, 32).
P2y and
P2u receptors are G protein coupled and mediate inositol trisphosphate
(IP3)-induced release of
Ca2+ from internal stores (2). By
initiating the release of small amounts of
Ca2+ near the plasma membrane,
P2y and
P2u occupation could lead to the
activation of Ca2+-dependent
outward currents. In the present study we found that ATP and the
P2y agonist 2-MeS-ATP activated
small-conductance K+ channels.
This observations suggests that
P2y receptors could mediate
ATP-dependent hyperpolarization. We cannot entirely rule out
involvement of P2u receptors, but
we found UTP to be less effective than 2-MeS-ATP in activating SK
channels. Nothing is known about the expression of
P2u receptors in colonic muscles, and the effects of UTP could have been mediated by nonspecific activation of P2y receptors. We
also found that the P2 receptor antagonist PPADS (27) blocked responses to ATP and 2-MeS-ATP, but we
view this observation with caution, because a recent study has
suggested that PPADS may nonselectively inhibit
IP3 receptors, rather than block
P2y receptors (42). It should be
clearly stated that the exact nature of the receptor(s) that mediates
the ATP responses we have observed is difficult to determine, since
highly selective antagonists for these receptors are not available.
ATP had no effect on currents in cells with use of the standard,
dialyzing configuration of whole cell recording. This finding suggests
that intracellular second messengers, such as
Ca2+ or other factors, are
required for ATP-induced activation of outward current. The cells were
dialyzed with solutions containing EGTA in these experiments, and it is
possible that changes in intracellular
Ca2+ are important in mediating
the response to ATP. When the perforated-patch technique, which
preserves intracellular signaling mechanisms (34), was used, ATP
activated a K+ conductance (i.e.,
the current was outward and the I-V
relationship of the conductance was well fit by the GHK equation) when
an extracellular solution containing
Mn2+ (to replace
Ca2+) was used. When standard
Ca2+-containing extracellular
solution was used, the currents generated by ATP were not well
described by the GHK equation, and inward tail currents were observed
on repolarization. Thus ATP may generate a mixed current consisting of
the K+ current elicited in
Mn2+ solution plus
Ca2+-dependent
Cl
or nonselective cation
conductances. The characteristics of these inward conductances are not
a subject of this study. The fact that a mixed current is generated on
perfusion of cells with ATP suggests that release of this substance
from nerves in situ must be directed at specialized populations of
receptors [i.e., specialized motor end plate regions, as
demonstrated morphologically in some purinergically innervated tissues
(17)]. Otherwise it would be difficult to explain how release of
ATP from extrinsic sympathetic nerves results in excitation (41)
whereas release of ATP from intrinsic inhibitory neurons results in
hyperpolarization and inhibition (20).
Binding of P2y receptors is
coupled via G proteins to activation of phospholipase C,
IP3 formation, and release of
Ca2+ from internal stores (15). We
suggest that this may be the major pathway of coupling between ATP
binding and activation of SK channels, because
1) ATP had no effect on cells
dialyzed with EGTA to buffer intracellular
Ca2+,
2) ATP activated an outward current
in cells in which influx of Ca2+
was blocked by replacement of external
Ca2+ with
Mn2+,
3) caffeine treatment also activated
SK independent of purine receptors, and
4) after an initial activation of SK
with caffeine, open probability waned, possibly from depletion of
sarcoplasmic reticulum Ca2+
stores. During this refractoriness, further addition of ATP failed to
activate SK channels. The fact that PPADS also blocked SK channel activation in response to ATP and 2-MeS-ATP may also support the notion
that IP3-dependent release of
Ca2+ is responsible for activation
of SK channels, since PPADS may block this mechanism (42).
It might seem odd that the effects of a compound reputed to be an
inhibitory neurotransmitter are mediated by the release of
Ca2+ in smooth muscle cells.
However, recent studies have shown that Ca2+ release from internal stores
can be highly directional and cause local increases in subsarcolemmal
Ca2+ concentration without
affecting global Ca2+ (33). A
number of Ca2+-dependent
conductances are expressed in smooth muscle cells (12), and the issue
of how Ca2+-dependent inhibitory
or excitatory responses might be preferentially activated should be
considered. One level of selectivity may result from the relative
Ca2+ sensitivity and voltage
dependence of inward and outward currents. For example, SK channels are
more sensitive to Ca2+ (7) than
most of the other conductances present. We found that SK channels were
activated at <100 nM Ca2+, even
at negative membrane potentials. Therefore, small increases in
Ca2+ near the membrane may
selectively activate SK channels, and agonists that release
focalized, small amounts of
Ca2+ may preferentially cause
activation of SK channels. Higher concentrations of
Ca2+, perhaps supplemented by
entry of Ca2+ or more potent
stimulation of Ca2+ release, may
facilitate nonselective cation channels and activate Ca2+-dependent
Cl
conductances and BK
channels (for review see Ref. 12). Opening of BK channels tends to be
constrained by the negative membrane potential range over which smooth
muscle cells operate, but this conductance appears to provide important
feedback regulation of membrane potential in some smooth muscles (8).
Activation of Cl
and
nonselective cation channels provides a depolarizing influence that is
reinforced by positive feedback as
Ca2+ enters via voltage-dependent
Ca2+ channels.
Ca2+-dependent conductances could
also be selectively regulated by the physical proximity of receptors
and channels. For example, the location of
P2y receptors near SK channels may
facilitate Ca2+-dependent
coupling, because local production of
IP3 might produce a
"sparklike" event that might affect nearby channels without significantly affecting Ca2+
levels near more distant channels. We were able to activate SK channels
within the pipette in the "on-cell" configuration by adding
purines to the external bath solution. This observation does not
support an extremely close obligatory association between P2y receptors and SK channels and
suggests that IP3 production resulting from P2y binding may
raise local Ca2+ within a diameter
of
1 µm.
Responses to ATP were sustained for several minutes in whole cell and
single channel experiments. Sustained hyperpolarization and inhibition
of spontaneous electrical activity have also been noted when murine
colonic muscles are exposed to exogenous ATP (45). If ATP-dependent
activation of SK channels is due to release of
Ca2+ from the sarcoplasmic
reticulum, then the sustained nature of these responses suggests that
ATP stimulation increases the rate of
Ca2+ release rather than causes a
massive dumping of the Ca2+
stores. A graded increase in the open probability of
Ca2+ release channels may
facilitate local increases in Ca2+
in the vicinity of SK channels without having an impact on global Ca2+. Graded release of
Ca2+ from intracellular stores has
been previously demonstrated by responses to caffeine. In the first
reported measurements of spontaneous transient outward currents
(STOCs), now considered a hallmark of
Ca2+ release from stores in smooth
muscles, caffeine had a differential response depending on dose: a high
concentration of caffeine caused a burst of STOCs and then quiescence;
a lower concentration caused a sustained increase in the frequency of
STOCs (3). These observations are now known to be due to the effects of
caffeine on ryanodine receptors (33). A high concentration of caffeine
greatly increases the open probability of ryanodine receptors and
"dumps" the Ca2+ stores.
After the stores are dumped, there are no release events and no STOCs.
A lower caffeine concentration increases the open probability of
ryanodine receptors to a lesser extent, and the stores are not rapidly
emptied. Thus the increase in the frequency of STOCs is sustained.
Something of this nature (i.e., graded Ca2+ release via an
IP3-dependent mechanism) may
explain the sustained effects of ATP on SK channels in murine colonic
cells.
We are unable to say with certainty that the 5.3-pS single channel
currents activated by ATP and 2-MeS-ATP are the basis for the whole
cell currents and hyperpolarization responses in intact muscles
activated by these agonists. We could not make clear recordings of such
small channels in "outside-out" patches, and therefore we could
not adequately test the effects of apamin on these channels. Even if
this experiment were practical, it may not be definitive, because
apamin reduced peak whole cell current by only about one-third. ATP may
activate more than a single type of
K+ conductance in whole cell
experiments, and it is also possible that apamin-sensitive and
-insensitive isoforms of SK channels could contribute to the whole cell
response to ATP. It would be very difficult to demonstrate the latter
simply by addition of apamin to the pipette solution. A tentative link
between the single channels and whole cell currents can be made
inferentially on the basis of the following observations:
1) the current activated by ATP was
partially blocked by apamin; 2)
apamin blocks some isoforms of SK channels (25);
3) whole cell currents were not resolved when cell interiors were dialyzed and buffered for
Ca2+;
4) SK channels are
Ca2+ dependent (7); and
5) the single channels activated by
ATP and Ca2+ had properties
consistent with SK channels. Other conductances, not manifest in our
single channel recordings, could also contribute to the whole cell
response. In the future, it will be interesting to test the hypothesis
that apamin-sensitive and -insensitive isoforms of SK channels are
expressed by GI muscles using Northern analysis and isoform-specific
cDNA probes.
 |
NOTE ADDED IN PROOF |
After acceptance of this paper, another paper showing activation of
small-conductance Ca2+-activated K+ channels in
response to purinergic agonists appeared in the literature [F. Vogalis
and R. K. Goyal. Activation of small conductance
Ca+-dependent K+ channels by purinergic
agonists in smooth muscle cells of the mouse ileum. J. Physiol.
(Lond.) 502: 497-508, 1977].
 |
ACKNOWLEDGEMENTS |
We are very grateful to Drs. Kenyon and Shuttleworth for critical
reading of the manuscript and helpful suggestions.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Program Project Grant DK-41315.
Address reprint requests to K. M. Sanders.
Received 24 March 1997; accepted in final form 28 July 1997.
 |
REFERENCES |
1.
Banks, B. E.,
C. Brown,
G. M. Burgess,
G. Burnstock,
M. Claret,
T. M. Cocks,
and
D. H. Jenkinson.
Apamin blocks certain neurotransmitter-induced increases in potassium permeability.
Nature
282:
415-417,
1979[Medline].
2.
Barnard, E. A.,
G. Burnstock,
and
T. E. Webb.
G protein-coupled receptors for ATP and other nucleotides: a new receptor family.
Trends Pharmacol. Sci.
15:
67-70,
1994[Medline].
3.
Benham, C. D.,
and
T. B. Bolton.
Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit.
J. Physiol. (Lond.)
381:
385-406,
1986[Abstract].
4.
Benham, C. D.,
T. B. Bolton,
R. J. Lang,
and
T. Takewaki.
Calcium-activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery.
J. Physiol. (Lond.)
371:
45-67,
1986[Abstract].
5.
Benham, C. D.,
and
R. W. Tsien.
A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle.
Nature
328:
275-278,
1987[Medline].
6.
Bennett, M. R.,
G. Burnstock,
and
M. Holman.
Transmission from intramural inhibitory nerves to the smooth muscle of the guinea-pig taenia coli.
J. Physiol. (Lond.)
182:
541-558,
1966[Medline].
7.
Blatz, A. L.,
and
K. L. Magleby.
Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle.
Nature
323:
718-720,
1986[Medline].
8.
Brayden, J. E.,
and
M. T. Nelson.
Regulation of arterial tone by activation of calcium-dependent potassium channels.
Science
256:
532-535,
1992[Medline].
9.
Bult, H.,
G. E. Boeckxstaens,
P. A. Pelckmans,
F. H. Jordaens,
Y. M. Van Maercke,
and
A. G. Herman.
Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter.
Nature
345:
346-347,
1990[Medline].
10.
Burnstock, G.,
and
C. Kennedy.
A dual function for adenosine 5'-triphosphate in the regulation of vascular tone. Excitatory cotransmitter with noradrenaline from perivascular nerves and locally released inhibitory intravascular agent.
Circ. Res.
58:
319-330,
1986[Abstract].
11.
Capiod, T.,
and
D. C. Ogden.
The properties of calcium-activated potassium ion channels in guinea-pig isolated hepatocytes.
J. Physiol. (Lond.)
409:
285-295,
1989[Abstract].
12.
Carl, A.,
H. K. Lee,
and
K. M. Sanders.
Regulation of ion channels in smooth muscles by calcium.
Am. J. Physiol.
271 (Cell Physiol. 40):
C9-C34,
1996[Abstract/Free Full Text].
13.
Dalziel, H. H.,
K. D. Thornbury,
S. M. Ward,
and
K. M. Sanders.
Involvement of nitric oxide synthetic pathway in inhibitory junction potentials in canine proximal colon.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G789-G792,
1991[Abstract/Free Full Text].
14.
Droogmans, G.,
G. Callewaert,
I. Declerck,
and
R. Casteels.
ATP-induced Ca2+ release and Cl
current in cultured smooth muscle cells from pig aorta.
J. Physiol. (Lond.)
440:
623-634,
1991[Abstract].
15.
Dubyak, G. R.,
and
C. el-Moatassim.
Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides.
Am. J. Physiol.
265 (Cell Physiol. 34):
C577-C606,
1993[Abstract/Free Full Text].
16.
Friel, D. D.
An ATP-sensitive conductance in single smooth muscle cells from the rat vas deferens.
J. Physiol. (Lond.)
401:
361-380,
1988[Abstract].
17.
Gabella, G.
The structural relations between nerve fibres and muscle cells in the urinary bladder of the rat.
J. Neurocytol.
24:
159-187,
1995[Medline].
18.
Goyal, R. K.,
S. Rattan,
and
S. I. Said.
VIP as a possible neurotransmitter of non-cholinergic non-adrenergic inhibitory neurons.
Nature
288:
378-380,
1980[Medline].
19.
He, X. D.,
and
R. K. Goyal.
Nitric oxide involvement in the peptide VIP-associated inhibitory junction potential in the guinea-pig ileum.
J. Physiol. (Lond.)
461:
485-499,
1993[Abstract].
20.
Hoyle, C. V. H.,
and
G. Burnstock.
Neuromuscular transmission in the gastrointestinal tract.
In: Handbook of Physiology. The Alimentary Canal. Motility and Circulation. Bethesda, MD: Am. Physiol. Soc., 1989, sect. 6, vol. I, p. 435-464.
21.
Hu, S. L.,
Y. Yamamoto,
and
C. Y. Kao.
The Ca2+-activated K+ channel and its functional roles in smooth muscle cells of guinea pig taenia coli.
J. Gen. Physiol.
94:
833-847,
1989[Abstract].
22.
Inoue, R.,
K. Kitamura,
and
H. Kuriyama.
Two Ca-dependent K-channels classified by the application of tetraethylammonium distribute to smooth muscle membranes of the rabbit portal vein.
Pflügers Arch.
405:
173-179,
1985[Medline].
23.
Keef, K. D.,
C. Du,
S. M. Ward,
B. McGregor,
and
K. M. Sanders.
Enteric inhibitory neural regulation of human colonic circular smooth muscle: role of nitric oxide.
Gastroenterology
105:
1009-1016,
1993[Medline].
24.
Koh, S. D.,
K. M. Sanders,
and
A. Carl.
Regulation of smooth muscle delayed rectifier K+ channels by protein kinase A.
Pflügers Arch.
432:
401-412,
1996[Medline].
25.
Kohler, M.,
B. Hirschberg,
C. T. Bond,
J. M. Kinzie,
N. V. Marrion,
J. Maylie,
and
J. P. Adelman.
Small-conductance, calcium-activated potassium channels from mammalian brain.
Science
273:
1709-1714,
1996[Abstract/Free Full Text].
26.
Komori, K.,
and
H. Suzuki.
Distribution and properties of excitatory and inhibitory junction potentials in circular muscle of the guinea-pig stomach.
J. Physiol. (Lond.)
370:
339-355,
1986[Abstract].
27.
Lambrecht, G.,
T. Friebe,
U. Grimm,
U. Windscheif,
E. Bungardt,
C. Hildebrandt,
H. G. Baumert,
G. Spatz-Kumbel,
and
E. Mutschler.
PPADS, a novel functionally selective antagonist of P2 purinoceptor-mediated responses.
Eur. J. Pharmacol.
217:
217-219,
1992[Medline].
28.
Li, C. G.,
and
M. J. Rand.
Evidence for a role of nitric oxide in the neurotransmitter system mediating relaxation of the rat anococcygeus muscle.
Clin. Exp. Pharmacol. Physiol.
16:
933-938,
1989[Medline].
29.
Li, C. G.,
and
M. J. Rand.
Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, non-cholinergic inhibitory transmission to smooth muscle of the rat gastric fundus.
Eur. J. Pharmacol.
191:
303-309,
1990[Medline].
30.
Maas, A. J.,
and
A. Den Hertog.
The effect of apamin on the smooth muscle cells of the guinea-pig taenia coli.
Eur. J. Pharmacol.
58:
151-156,
1979[Medline].
31.
McConalogue, K.,
D. J. Lyster,
and
J. B. Furness.
Electrophysiological analysis of the actions of pituitary adenylyl cyclase activating peptide in the taenia of the guinea-pig caecum.
Naunyn Schmiedebergs Arch. Pharmacol.
352:
538-544,
1995[Medline].
32.
Molleman, A.,
A. Nelemans,
and
A. Den Hertog.
P2-purinoreceptor-mediated membrane currents in DDT1MF-2 smooth muscle cells.
Eur. J. Pharmacol.
169:
167-174,
1989[Medline].
33.
Nelson, M. T.,
H. Cheng,
M. Rubart,
L. F. Santana,
A. D. Bonev,
H. J. Knot,
and
W. J. Lederer.
Relaxation of arterial smooth muscle by calcium sparks.
Science
270:
633-637,
1995[Abstract].
34.
Rae, J.,
K. Cooper,
P. Gates,
and
M. Watsky.
Low access resistance perforated patch recordings using amphotericin B.
J. Neurosci. Methods
37:
15-26,
1991[Medline].
35.
Sanders, K. M.,
and
S. M. Ward.
Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission.
Am. J. Physiol.
262 (Gastrointest. Liver Physiol. 25):
G379-G392,
1992[Abstract/Free Full Text].
36.
Shuttleworth, C. W. R.,
S. B. Conlon,
and
K. M. Sanders.
Regulation of citrulline recycling in nitric oxide-dependent neurotransmission in murine proximal colon.
Br. J. Pharmacol.
120:
707-713,
1997[Abstract].
37.
Smith, T. K.,
J. B. Reed,
and
K. M. Sanders.
Electrical pacemakers of canine proximal colon are functionally innervated by inhibitory motor neurons.
Am. J. Physiol.
256 (Cell Physiol. 25):
C466-C477,
1989[Abstract/Free Full Text].
38.
Stark, M. E.,
A. J. Bauer,
M. G. Sarr,
and
J. H. Szurszewski.
Nitric oxide mediates inhibitory nerve input in human and canine jejunum.
Gastroenterology
104:
398-409,
1993[Medline].
39.
Thornbury, K. D.,
S. M. Ward,
H. H. Dalziel,
A. Carl,
D. P. Westfall,
and
K. M. Sanders.
Nitric oxide and nitrosocysteine mimic nonadrenergic, noncholinergic hyperpolarization in canine proximal colon.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G553-G557,
1991[Abstract/Free Full Text].
40.
Tomita, T.
Conductance change during the inhibitory potential in the guinea-pig taenia coli.
J. Physiol. (Lond.)
225:
693-703,
1972[Medline].
41.
Venkova, K.,
and
J. Krier.
Stimulation of lumbar sympathetic nerves evokes contractions of cat colon circular muscle mediated by ATP and noradrenaline.
Br. J. Pharmacol.
110:
1260-1270,
1993[Abstract].
42.
Vigne, P.,
P. Pacaud,
V. Urbach,
E. Feolde,
J. P. Breittmayer,
and
C. Frelin.
The effect of PPADS as an antagonist of inositol (1,4,5)trisphosphate induced intracellular calcium mobilization.
Br. J. Pharmacol.
119:
360-364,
1996[Abstract].
43.
Vladimirova, I. A.,
and
M. F. Shuba.
Effect of strychnine, hydrastine and apamin on synaptic transmission in smooth muscle cells.
Neirofiziologiia
10:
295-299,
1978[Medline].
44.
Von Kugelgen, I.,
and
K. Starke.
Noradrenaline-ATP co-transmission in the sympathetic nervous system.
Trends Pharmacol. Sci.
12:
319-324,
1991[Medline].
45.
Ward, S. M.,
and
V. M. Jackson.
Identification of the neurotransmitter mediating the fast inhibitory junction potential in the murine colon (Abstract).
Gastroenterology
112:
A850,
1996.
46.
Weir, S. W.,
and
A. H. Weston.
The effects of BRL 34915 and nicorandil on electrical and mechanical activity and on 86Rb efflux in rat blood vessels.
Br. J. Pharmacol.
88:
121-128,
1986[Abstract].
47.
Winquist, R. J.,
L. A. Heaney,
A. A. Wallace,
E. P. Baskin,
R. B. Stein,
M. L. Garcia,
and
G. J. Kaczorowski.
Glyburide blocks the relaxation response to BRL 34915 (cromakalim), minoxidil sulfate and diazoxide in vascular smooth muscle.
J. Pharmacol. Exp. Ther.
248:
149-156,
1989[Abstract].
48.
Yamanaka, K.,
K. Furukawa,
and
K. Kitamura.
The different mechanisms of action of nicorandil and adenosine triphosphate on potassium channels of circular smooth muscle of the guinea-pig small intestine.
Naunyn Schmiedebergs Arch. Pharmacol.
331:
96-103,
1985[Medline].
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