Effect of nitric oxide on calcium-activated potassium channels
in colonic smooth muscle of rabbits
Gang
Lu1,
Bruno
Mazet1,
Michael G.
Sarr2, and
Joseph H.
Szurszewski1
1 Department of Physiology and
Biophysics, and 2 Department of
Surgery, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
55905
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ABSTRACT |
Nitric oxide (NO)
hyperpolarizes intestinal smooth muscle cells. This study was designed
to determine the mechanism whereby NO activates
KCa channels of circular smooth
muscle of the rabbit colon. Transmural biopsies of the rabbit colon
were stained for NADPH-diaphorase. Freshly dispersed circular smooth
muscle cells were studied in the whole cell configuration, as well as
in on-cell and excised inside-out patch recording configurations, while
KCa current and the
activity of KCa channels,
respectively, were monitored. NADPH-diaphorase-positive nerve fibers
were found in both muscle layers. NO (1%) increased whole cell net
outward current by 79% and hyperpolarized resting membrane voltage
from
59 to
73 mV (n = 8 cells, P < 0.01). In the on-cell
patch recording configuration, NO (0.5% or 1%) in the bath increased
NPo of
KCa channels; charybdotoxin (125 nM) in the pipette solution blocked this effect. In the excised inside-out patch recording configuration, NO (1%) had no effect on
NPo of
KCa channels. In the on-cell patch
recording configuration, methylene blue (1 µM) or cystamine (5 mM) in
the bath solution decreased the effect of NO (1%) on
NPo of
KCa channels.
NPo was increased
by 8-bromo-cGMP (8-BrcGMP; 1 mM), a cGMP analog, and zaprinast (100 µM), an inhibitor of cGMP phosphodiesterase. These data suggest that
NO increased whole cell outward K+
current by activating KCa channels
through a cGMP pathway.
guanosine 3',5'-cyclic monophosphate; NADPH diaphorase; dispersed smooth muscle cells
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INTRODUCTION |
STIMULATION of nonadrenergic, noncholinergic (NANC)
nerves evokes a membrane hyperpolarization in circular smooth muscle
cells of the gastrointestinal (GI) tract. The hyperpolarizing response is referred to as the inhibitory junction potential (IJP). Recent studies have suggested that nitric oxide (NO) is a NANC inhibitory neurotransmitter that mediates at least part of the IJP (26, 31) in
canine and human jejunum (30), canine colon (8), and esophageal smooth
muscle (7, 10, 21).
In smooth muscle cells of the opossum esophagus, NO increases whole
cell outward current via a cGMP-dependent pathway (21). In the canine
colon, cGMP analogs mimic the effect of NO by hyperpolarizing smooth
muscle cells and inhibiting phasic contractions (34, 38). Preliminary
data in canine colonic myocytes indicate that NO increases the open
probability
(NPo) of
K+ channels that were assumed
to be Ca2+-activated K+
(KCa) channels (34). The
increase in NPo
of these large-conductance K+
channels in response to NO has been recently confirmed (19). The
purpose of this study was to test the hypothesis that NO increases NPo of
KCa channels in rabbit colonic
myocytes and that this effect was mediated through a cGMP-dependent
pathway.
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MATERIALS AND METHODS |
Adult New Zealand White rabbits (2-3 kg) of either sex were
euthanized by a lethal dose of intravenous pentobarbital sodium (100 mg/kg). The use of rabbits and the method of euthanasia were approved
by the Mayo Animal Care and Use Committee. A section of 5-8 cm of
the distal colon was removed and placed in a cold (4°C)
physiological salt solution (PSS; Table 1).
NADPH-Diaphorase Staining
The distribution of NADPH-diaphorase (NADPH-d)-positive nerve fibers in
the muscularis externa was studied in whole mounts of colonic tissue.
Colonic segments were opened along the antimesenteric border, and the
mucosa was washed with PSS. Pieces (2 × 2 cm) of colon were
pinned out with the mucosal side down in a glass dish, and
fixed for 4-6 h at 4°C in 4% paraformaldehyde in 0.1 M
phosphate buffer pH 7.4 (PB). The tissues were rinsed first in PB
(three 20-min periods), then in 0.1 M Tris buffer containing 0.3%
Triton X-100, pH 7.4 (TTB) for 10 min. The mucosa was removed by
dissection and discarded. The tissues were incubated in TTB containing
1 mg/ml
-NADPH and 0.2 mg/ml nitro blue tetrazolium for 15-20
min at 35°C. The reaction was stopped by rinsing the tissue with
cold PB. Control experiments using an incubation medium without
substrate (
-NADPH) showed no staining. The tissues were dehydrated
in ethanol and cleared in Histoclear (National Diagnostics) before
mounting in Cytoseal (Stephens Scientific) for examination under a
light microscope.
Cell Isolation Technique
Circular smooth muscle cells of the distal colon were isolated using a
modification of the technique described by Benham et al. (2). The
circular smooth muscle layer was cut into strips 2-3 mm wide and
5-10 mm long. The muscle strips were incubated for 25 min at
37°C in PSS containing 98 U/ml collagenase type II (Worthington
Biochemical, Freehold, NJ), 1 mg/ml soybean trypsin inhibitor, and 2 mg/ml bovine serum albumin (Sigma Chemical, St. Louis, MO) while the
solution was agitated continuously at 100 cycles/min with a circulator
shaking incubator (model 1201-00; Cole-Palmer Instrument, Chicago,
IL). Single relaxed smooth muscle cells were transferred to fresh PSS
and stored at 4°C for up to 8 h.
Patch-Clamp Techniques
Freshly dispersed cells were allowed to settle on the flat bottom of a
500-µl glass chamber mounted on an inverted microscope stage. The
chamber was superfused (2 ml/min) with oxygenated bicarbonated PSS at
room temperature (21-23°C). Patch pipettes were made from type
KG12 glass (Friedrich & Dimmock, Millville, NJ) for recording whole
cell currents and from type 7052 glass (Garner Glass, Claremont, CA)
for recording single-channel currents. Glass pipettes were pulled on a
model P-87 Flaming/Brown micropipette puller (Sutter Instrument,
Novato, CA), coated with Sylgard 184 (Dow Corning, Midland, WI), and
fire-polished to a final resistance of ~4-5 M
when filled
with PSS. Pipette-membrane seal resistances ranged from 10 to 15 G
.
All recordings in the whole cell, on-cell patch, and excised inside-out
patch recording configurations were obtained using standard patch-clamp
techniques (16). Liquid-junction potentials
(Vj) of pipette
tips ranged from 9 to 11 mV when whole cell recording solution (WCRS;
Table 1) and PSS were used as the pipette and bath solutions,
respectively. In the whole cell recording configuration, the membrane
potential (Vm)
was assumed to be equal to
Vm = Vcom
Vj, where
Vcom is the
command voltage. In the on-cell patch configuration,
Vp = Vr = (Vcom
Vj), where Vp is the
membrane voltage of the patch and
Vr is the resting voltage of the patch. In the inside-out patch configuration,
Vm =
Vcom.
Recordings of single-channel currents and whole cell currents were made
with an Axopatch patch-clamp amplifier (Axon Instruments, Foster, CA).
Whole cell currents were sampled at 2 kHz and filtered at
1 kHz. Single-channel data were sampled at 2 kHz and filtered at 500 Hz. The data were analyzed using pClamp software (Axon Instruments).
Open probability
(Po) was
estimated using the following equation (25)
where
T is the duration of the test pulse,
N the maximum number of channels
observed during a depolarizing test pulse, and ti the time
spent with i = 1,2,...N channels open.
Because the majority of patches contained more than one channel,
channel activity was quantified by evaluating the product parameter.
Solutions and Drugs
The solutions and drugs used in the NADPH-d staining experiments were
obtained from Sigma. The composition of the solutions used for
patch-clamp recordings is given in Table 1. NO-containing solutions
were prepared by a method similar to the one described by Palmer et al.
(22). Briefly, a gas bulb sealed with a rubber injection septum was
filled at atmospheric pressure with NO gas (Applied Gas Technology, La
Porte, TX). An appropriate volume (500 or 1,000 µl) was removed with
a syringe and injected into another gas bulb filled with 100 ml of the
appropriate solution (Table 1), depending on the experimental
conditions [PSS, WCRS, or inside-out patch recording solution
(IPRS)]. The solutions were deoxygenated previously by gassing
with He for 2 h, to give stock solutions of 0.5% and 1% NO (vol/vol),
respectively. NO solution (1 ml) of the desired concentration was
infused directly into the recording chamber over ~25 s using a
syringe and needle. The concentration of NO in the infused volume was
reduced by an unknown amount before reaching the cells, because NO is
unstable in oxygenated solution. Methylene blue and cystamine (Sigma)
were dissolved in PSS. Zaprinast (gift from Rhrone-Poulenc Rohrer) was
first dissolved in 0.1 M NaOH and then added to PSS to give the final
concentration. Charybdotoxin (ChTX; Alomone Laboratories, Jerusalem,
Israel) was dissolved in PSS and stored at 0°C until the day of the
experiment.
Statistical Analysis
Data are presented as means ± SD. Comparisons of measurements
between groups were made using Student's
t-test for paired or unpaired data,
depending on the experimental design.
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RESULTS |
Distribution of NADPH-d Staining
Numerous NADPH-d reactive nerve cell bodies were present in myenteric
(Fig. 1) and submucosal ganglia (not
shown). NADPH-d-positive nerve fibers were found in nerve bundles
connecting enteric ganglia (Fig.
1A) and in the longitudinal (Fig.
1B) and circular (Fig. 1C) muscle layers. Single
NADPH-d-positive nerve fibers coursed parallel to smooth muscle cells;
their density was greater in the circular muscle layer compared with
the longitudinal muscle layer (Fig. 1,
C vs.
B).

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Fig. 1.
Distribution of NADPH-diaphorase (NADPH-d) staining in the rabbit
colon. NADPH-d-positive reactivity was found in nerve cell bodies in
myenteric ganglia (A) and in nerve
fibers in longitudinal (B) and
circular (C) muscle layers. Note
that the density of NADPH-d-positive nerve fibers was greater in the
circular muscle layer.
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Characteristics of
KCa Channels
General observations. Eighty-eight
circular smooth muscle cells (obtained from 28 rabbits) were used in
this study. The membrane potential and membrane capacitance recorded in
zero current clamp mode were
59 ± 5 mV and 79 ± 8 pF,
respectively. In most cells, inward current was not detected under
control conditions.
Whole cell outward current. An example
of the whole cell outward current typically recorded in this study from
a colonic circular smooth muscle cell when the cell was bathed in 6 mM
K+ (PSS) and the recording pipette
solution contained 150 mM K+
(WCRS, Table 1) is shown in Fig.
2A. The
outward current was well maintained throughout the test pulses. The
mean current-voltage relationship for the outward current for four
cells also is shown in Fig. 2C. A
significant fraction of the outward current was sensitive to block by
ChTX, a selective blocker of KCa
channels (Fig. 2B). Addition of ChTX
(125 nM) to the bathing solution decreased outward current by 47 ± 3% (n = 4 cells,
P < 0.01) at
Vcom = +40 mV
(Fig. 2C). Similarly, a reduction in
the concentration of free Ca2+ in the bath solution to 0 mM
significantly decreased the outward current by 48 ± 3%
(n = 4 cells,
P < 0.01) at
Vcom = +40 mV
(Fig. 3). The addition of
tetraethylammonium (TEA, 2 mM) to the bath solution
reduced outward current by 49 ± 6%
(n = 4 cells,
P < 0.01; Fig.
4, B and
D); 20 mM TEA reduced outward
current by 98 ± 1% (n = 4 cells,
P < 0.01) (Fig. 4,
C and
D). Taken together, these data
suggest that a Ca2+-activated
TEA-sensitive K+ current was a
significant fraction of the outward current. Previous studies have
shown that the KCa current in
small and large intestinal smooth muscle is sensitive to TEA (2, 5,
29). Although not examined, the outward current that remained when
ChTX, a low concentration of TEA, or a reduced extracellular free
Ca2+ solution was present in the
bath most likely was a delayed rectifying K+ current, as previously
described for small intestinal circular smooth muscle cells (11).

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Fig. 2.
Whole cell outward current in absence
(A) and presence
(B) of charybdotoxin (125 nM).
Membrane voltage was stepped for 200 ms from 60 mV to voltages
ranging from 80 to +40 mV, and the cell was held at 60 mV
between test pulses. Each data point of the current-voltage plot
(C) represents the mean ± SD
(n = 4) of the peak current observed
at the indicated voltages. ** Significant
(P < 0.01) difference from control.
Bath solution contained 6 mM K+
and 1 mM Ca2+ (PSS, Table 1), and
pipette solution contained 150 mM
K+ and 2 µM free
Ca2+ (whole cell recording
solution, WCRS; Table 1). Charybdotoxin was added to the bath
solution.
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Fig. 3.
Effect of reducing free Ca2+
concentration in the bath solution on outward whole cell current. Each
data point of the current-voltage plot
(C) represents mean ± SD
(n = 4) of the peak current at the
indicated voltages. ** Significant
(P < 0.01) difference from 2.5 mM
Ca2+ in bath. The recording pipette solution contained 150 mM K+, 2 µM free
Ca2+ (WCRS, Table 1). Bath
solution in A was 6 mM
K+ and 1 mM
Ca2+ (PSS, Table 1); in
B the bath solution was 6 mM
K+ and 0 mM
free-Ca2+ PSS.
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Fig. 4.
Outward whole cell current without (control;
A) and with 2 mM
(B) and 20 mM
(C) tetraethylammonium (TEA) added
to the bath solution. Each data point of the current-voltage plot
(D) represents mean ± SD
(n = 4) of the peak current observed
at the indicated voltage. **Significant
(P < 0.01) difference from control.
Bath and pipette solutions were PSS and WCRS, respectively (Table
1).
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Single-channel current. The
current-voltage relationship of single-channel currents in excised
inside-out patches was measured when
1) the recording pipette solution
contained 150 mM K+ plus 2 µM
free Ca2+ and the bath solution
contained 150 mM K+ plus 200 µM
free Ca2+; and
2) when the recording pipette
contained 150 mM K+ plus 2 µM
free Ca2+ and the bath solution
contained 6 mM K+ plus 1 mM
Ca2+. Stepping the voltage
(Vm) across the
patch to hyperpolarizing potentials from
40 to +40 mV increased
NPo and increased
single-channel current amplitude. The current-voltage relationships
observed are shown in Fig. 5. The mean
single-channel conductance in symmetrical KCl was 226 ± 12 pS
(n = 4) cells (Fig. 5). It can be seen
in Fig. 5 that the observed (symbols) current-voltage relationships agreed well with the expected (solid lines) relationship as calculated from the Goldman-Hodgkin-Katz equation.

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Fig. 5.
Current-voltage relationship of single-channel current in excised
inside-out patches of rabbit colonic circular smooth muscle cells.
Recordings of single-channel current were made at steady state at the
command voltages indicated. The mean ± SD
(n = 4) single-channel conductance was
226 ± 12 pS when symmetrical (150 mM) KCl solutions were used. The
continuous lines are the expected relationships calculated from the
Goldman-Hodgkin-Katz equation (15, 17). For additional details, see
text.
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The activity of KCa channels
observed in the present study in excised inside-out patches was
Ca2+ dependent. Decreasing the free
Ca2+ concentration in the bath
solution from 2 to 0.2 µM in symmetrical (150/150 mM) KCl solutions
decreased NPo
from 0.08 ± 0.01 to 0.03 ± 0.01 (Vm = +40 mV;
n = 4 cells,
P < 0.01). An example of the effect
of decreasing free Ca2+ on
NPo is shown in
Fig. 6.

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Fig. 6.
Effect of decreasing the concentration of free
Ca2+, from 5 × 10 4 M
(A) to 5 × 10 7 M
(B) in the bath solution on
single-channel activity recorded from an excised inside-out patch.
Recording pipette solution contained inside-out patch recording
solution (IPRS) with 2 × 10 6 M free
Ca2+ (Table 1); bath solution was
IPRS (Table 1) with 2 × 10 7 M
Ca2+. Dashes to the
left indicate open state; outward
current is upward. Vm, membrane potential.
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The NPo of
KCa channels in on-cell patches
was sensitive to ChTX and TEA. The addition of ChTX (125 nM) to the
recording pipette solution (PSS, Table 1) decreased
NPo from 0.08 ± 0.01 to 0.00 ± 0.00 (n = 4 cells). Addition of TEA (2 mM) to the recording pipette solution (PSS,
Table 1) decreased
NPo from 0.08 ± 0.01 to 0.00 ± 0.00 (n = 4 cells, P < 0.01). An example of the
effects of ChTX and TEA on
NPo is shown in
Figs. 7 and 8,
respectively. These data and those reported above suggested that the
observed single channels were KCa
channels.

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Fig. 7.
Effect of adding 125 nM charybdotoxin
(B) to the recording pipette
solution on single-channel activity recorded from an on-cell patch;
A is control. Recording pipette and
bath solutions were PSS (Table 1). Dashes to the
left indicate open state; outward
current is upward.
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Fig. 8.
Effect of adding 2 mM TEA (B) to the
bath solution on single-channel activity recorded from an on-cell
patch; A is control. Recording pipette
and bath solutions were PSS (Table 1). Dashes to the
left indicate open state; outward
current is upward.
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Effect of NO on Whole Cell Outward
Current
In the whole cell recording configuration (PSS in the bath and WCRS in
the recording pipette, Table 1), NO (1%) increased whole cell outward
current in eight of eight cells tested. The maximum NO-induced increase
in whole cell outward current lasted from 20 to 90 s in the different
cells tested and thereafter declined to control levels by 3 min.
Infusion of the vehicle had no effect on whole cell current
(n = 8 cells). The mean maximum
increase in the amplitude of the outward current caused by NO (1%) was 79 ± 28% (at +40 mV, n = 8 cells, P < 0.001). NO (1%)
also significantly shifted the membrane potential from
59.5 ± 5 to
73 ± 6 mV
(P < 0.05, n = 8 cells). An example of the effect
of NO on whole cell outward current and the effect of NO (1%) on the
current-voltage relationship for the outward current in eight cells is
shown in Fig. 9. Note that NO increased the
outward current at all voltages tested. When the difference current was
plotted (data not shown), the current reversed at a mean voltage of
73.5 mV (n = 8), suggesting a
specific effect of NO on K+
conductance. When ChTX (125 nM) was present in bath, NO (1%) had no
effect on the whole cell current (236.3 ± 12.5 vs. 237.3 ± 25.6 pS,
Vcom = +40 mV;
n = 4 cells,
P > 0.05).

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Fig. 9.
Increase in whole cell outward current due to NO (1%).
A: control. B: NO added to the
bath solution increased outward whole cell current.
C: mean current-voltage plot in
absence and presence of NO in the bath solution. * P < 0.05, ** P < 0.01, ***P < 0.001 vs. control.
Bath and pipette solutions were PSS and WCRS, respectively (Table 1).
Each data point is the mean ± SD
(n = 8).
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Effect of NO on single channels of on-cell
patches. Single-channel activity was recorded at
several command voltages. At each command voltage, 10 s of activity
were digitized at a sampling rate of 2 kHz. Although recordings were
made at command voltages ranging from
20 to
140 mV before
and during the addition of NO (0.5% or 1%) to the bath, channel
activity of on-cell patches at a command voltage of
100 mV was
of particular interest, because KCa channels are expected to be
fully opened at
100 mV. In all experiments PSS (Table 1) was
present in the bath and recording pipette. An example of the effect of
NO (1%) on channel activity at a command voltage of
100 mV is
shown in Fig. 10. Channel activity increased in the presence of NO, revealing the presence of many channels in the patch. Single-channel current amplitude was not affected by NO. When NO (1%) solution was used,
NPo at a command voltage of
100 mV increased significantly from 0.11 ± 0.04 to 0.71 ± 0.48 (n = 4, P < 0.01). When a 0.5% NO solution
was used, NPo
increased significantly from 0.12 ± 0.06 to 0.21 ± 0.12 (n = 4 cells,
P < 0.01). Infusion of the same
volume of deoxygenated PSS used to dissolve NO had no effect on
NPo. The addition
of ChTX to the recording pipette solution blocked the effect of NO (1%
and 0.5%). In four cells, the addition of ChTX (125 nM) significantly decreased NPo
from 0.13 ± 0.07 to 0.00 ± 0.00 (P < 0.05) at a command voltage of
100 mV. When NO (1%) was added to the bath solution and the
pipette solution contained ChTX, no channel activity was observed
(n = 4 of 4 cells).

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Fig. 10.
Effect of NO (1%; B) on
single-channel activity obtained from an on-cell patch vs. control
(A). Dashes to the
left indicate open state; outward
current is upward. At least 5 channels were activated when NO was
present. Recording pipette and bath solutions were PSS (Table 1).
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Inhibitors of guanylate cyclase activity also blocked the effect of NO
on single-channel activity. When added to the bath solution for 3 min,
methylene blue (1 µM, n = 4 cells) and cystamine (5 mM, n = 4 cells), tested separately, had no significant
(P > 0.05) effect on
NPo of
cell-attached patches over the range of command voltages used
(
20 to
140 mV). Both inhibitors, however, significantly
decreased the effect of NO (1%) on
NPo over the same
range of command voltages (Fig. 11).

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Fig. 11.
Effect of separately adding methylene blue (1 µM;
B) and cystamine (5 mM;
A) to the bath solution on NO-evoked
increase in NPo.
Recordings obtained from two different on-cell patches. Pipette and
bath solution were PSS (Table 1).
* P < 0.05 and
** P < 0.01 compared with 1%
NO.
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Zaprinast (100 µM), a selective cGMP phosphodiesterase inhibitor,
added to the bath solution significantly increased
NPo at a command
voltage of
100 mV from 0.07 ± 0.04 to 0.13 ± 0.07 (n = 4 cells,
P < 0.05). The addition of NO
(0.5%) in the presence of zaprinast further significantly increased
NPo to 0.43 ± 0.07 (n = 4 cells,
P < 0.05).
The addition of 8-BrcGMP (1 mM) to the bath solution significantly
increased NPo at
a command voltage of
100 mV from 0.08 ± 0.04 to 0.41 ± 0.19 (n = 5 cells,
P < 0.01). The effect of NO on
NPo in the
presence of 8-BrcGMP was not tested.
Effect of NO on single channels of excised inside-out
patches. In these experiments, IPRS with 20 µM free
Ca2+ (Table 1) was present in both
the recording pipette and bath. NO (1% in IPRS solution) at a command
voltage of
40 mV had no effect on
NPo. In four
cells tested, NPo
was 0.12 ± 0.01 in the absence of NO and 0.11 ± 0.02 in the
presence of NO (P > 0.05). An
example of the lack of effect of NO on single-channel activity of an
excised inside-out patch is shown in Fig.
12. Similar results were obtained at a
Vm of
100
mV.

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Fig. 12.
Lack of effect of NO (1%; B) on
single-channel activity recorded from an excised inside-out patch vs.
control (A). NO was added to the
bath. Dashes to the left indicate open
state; outward current is upward. Bath and recording pipette solutions
were IPRS (Table 1).
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DISCUSSION |
The present study was designed to determine the effect of NO on whole
cell outward current and on single-channel activity in circular smooth
muscle of the rabbit colon as well as whether cGMP might be important
in mediating the effect of NO. Our data show that NO increased
KCa current by increasing the open
probability of KCa channels and
that this effect was mediated at least in part via a cGMP pathway.
A significant fraction of the whole cell outward current observed under
control conditions had many of the features of a
KCa current. It was
Ca2+ sensitive and blocked by ChTX, a specific inhibitor of
KCa current (13, 20). Also, the
activation voltage of the KCa
current observed in the present study was similar to that for the
KCa current observed in canine
pyloric and colonic smooth muscle (6, 28, 35). Similar features of
KCa current have been described previously for a variety of smooth muscles (33, 35, 36). A significant
fraction of the outward current observed in the present study was TEA
sensitive in accordance with previous observations made in different
types of smooth muscle (2, 4, 5, 28, 29). Unlike the results obtained
in smooth muscle cells of the opossum esophagus (21), however, the
KCa current observed in rabbit
colonic circular smooth muscle cells did not inactivate with pulses of
300-ms duration.
The single-channel activity observed in the present study had
properties similar to those attributable to
KCa channels found in smooth
muscle cells of the airway, bovine aorta, and myometrium (12, 23, 32,
36). The open probability of this channel-type was voltage sensitive
and Ca2+ sensitive, and ChTX decreased the open
probability. The single-channel conductance was 226 pS in symmetrical
(150 mM) K+ solutions. This large conductance value is
similar to conductance values found for
KCa channels in other GI smooth
muscles (2, 5). Thus the data of the present study strongly support the notion that the bulk of the whole cell outward current was a
KCa current.
NO increased the magnitude of the
KCa current and increased the open
probability of the single channels that carried this current. These
effects of NO were concentration dependent. The increase in the
KCa current was accompanied by
hyperpolarization of the membrane with a mean shift in the membrane
voltage of 14 mV. The effect of exogenous application of NO to increase
the open probability of KCa
channels in the on-cell patch recording configuration by three- to
sixfold confirms and extends preliminary data obtained in GI smooth
muscle (34). It is noteworthy that NO did not affect single-channel
conductance. In the excised inside-out patch recording configuration,
NO had no effect on open probability. This also has been reported in
canine colonic myocytes (19). This latter finding strongly suggests
that in circular smooth muscle of the rabbit and canine colon, NO had
no direct effect on KCa channels.
This is in contrast to vascular smooth muscle where NO can directly
activate these channels (3), but it does not exclude the involvement of
another pathway to modify channel activity. There is a report that
small-conductance K+ channel
activity in excised inside-out patches of canine colonic myocytes is
directly increased by NO (19). We have not observed these particular
channels in rabbit colonic myocytes.
The intracellular site of action of NO most likely was the soluble form
of guanylate cyclase (9). Several studies suggest a role for cGMP in
the inhibitory effects of NO in GI smooth muscle (7, 21, 38). In the
present study, application of 8-BrcGMP, a membrane-permeable analog of
cGMP, increased the open probability of
KCa channels in the on-cell patch
recording configuration. Conversely, methylene blue and cystamine, both
inhibitors of guanylate cyclase activity, decreased open probability,
and zaprinast potentiated it, as would be expected if the NO-induced
activation of these channels was mediated through a cGMP pathway.
However, the NO-induced increase in open probability was not entirely
abolished in the presence of inhibitors. It could be argued that the
particulate form of guanylate cyclase also may have mediated the effect
of NO because methylene blue is a specific inhibitor of the soluble form (18, 37). However, cystamine in fact is an inhibitor of both forms
of the enzyme and is more selective for the particulate form (24). Thus
it is more likely that the effect of NO on
KCa channels was also mediated in
part via a nondirect cGMP-independent pathway.
It is worth mentioning that the inhibitors of guanylate cyclase
(methylene blue and cystamine) did not by themselves affect the open
probability of KCa channels in
control conditions. These electrophysiological results are in agreement
with the results obtained by immunohistochemistry in circular smooth
muscle of the canine colon in which it was shown that there is no
ongoing production of cGMP in unstimulated GI smooth muscle cells (27). However, in the present study the phosphodiesterase inhibitor zaprinast
increased the activity of KCa
channels. This is in contrast to circular smooth muscle cells of the
canine colon where immunohistochemical methods failed to show ongoing
production of cGMP when inhibitors of phosphodiesterase were present
(27). This difference between canine and rabbit colon may be due to
species differences and/or to different forms of
phosphodiesterase (1). It is also possible that the resting level of
cGMP in canine colonic circular smooth muscle was below the threshold
of detection of the anti-cGMP antibody used (27).
The present results implicating NO in the rabbit colon were further
supported by our findings with immunohistochemical staining for NADPH-d
in whole mounts of colonic tissue. NADPH-d-positive nerve fibers were
evident in nerve bundles in the circular (and longitudinal) muscle
layer as well as in the myenteric plexus. These nerve fibers coursed
parallel to smooth muscle cells. Although these findings do not
directly implicate these nerves in control of smooth muscle cell
function, the presence of NADPH-d-positive nerves within the muscle
layer supports our electrophysiological findings concerning the effects
of NO on isolated smooth muscle cells.
In conclusion, the results of the present study show that NO increased
an outward KCa-current in rabbit
colonic circular smooth muscle and that this effect was mediated at
least in part through a cGMP pathway. Whether the
KCa channels were activated
directly by cGMP (14) or by cGMP-dependent protein kinases remains to be determined. NO also appeared to act through a cGMP-independent pathway. This latter effect, however, was not due to a direct action of
NO on KCa channels.
 |
ACKNOWLEDGEMENTS |
We thank Jan Applequist for secretarial assistance and Gary Stoltz
and Philip Schmalz for technical assistance.
 |
FOOTNOTES |
This work was supported in part by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-17238 (J. H. Szurszewski) and
DK-39337 (M. G. Sarr) and the Mayo Foundation.
Address for reprint requests: J. H. Szurszewski, Department of
Physiology and Biophysics, Mayo Clinic and Mayo Foundation, 200 First
St. SW, Rochester, MN 55905.
Received 11 June 1997; accepted in final form 6 January 1998.
 |
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