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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Composition of solutions

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 beta -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 (beta -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 MOmega when filled with PSS. Pipette-membrane seal resistances ranged from 10 to 15 GOmega . 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)
<IT>P</IT><SUB>o</SUB> = <FR><NU>1</NU><DE><IT>TN</IT></DE></FR> <LIM><OP>∑</OP><LL><IT>i</IT>=1</LL><UL><IT>n</IT></UL></LIM> <IT>t<SUB>i</SUB>i</IT>
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.
<IT>NP</IT><SUB>o</SUB> = <FR><NU><LIM><OP>∑</OP><LL><IT>i</IT>=1</LL><UL><IT>n</IT></UL></LIM> <IT>t<SUB>i</SUB>i</IT></NU><DE><IT>T</IT></DE></FR>

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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).


View larger version (81K):
[in this window]
[in a new window]
 
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.

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).


View larger version (27K):
[in this window]
[in a new window]
 


View larger version (16K):
[in this window]
[in a new window]
 
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.


View larger version (29K):
[in this window]
[in a new window]
 


View larger version (16K):
[in this window]
[in a new window]
 
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.


View larger version (23K):
[in this window]
[in a new window]
 


View larger version (19K):
[in this window]
[in a new window]
 
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).

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.


View larger version (15K):
[in this window]
[in a new window]
 
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.

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.


View larger version (12K):
[in this window]
[in a new window]
 
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.

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.


View larger version (12K):
[in this window]
[in a new window]
 
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.


View larger version (7K):
[in this window]
[in a new window]
 
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.

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).


View larger version (25K):
[in this window]
[in a new window]
 


View larger version (18K):
[in this window]
[in a new window]
 
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).

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).


View larger version (16K):
[in this window]
[in a new window]
 
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).

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).


View larger version (14K):
[in this window]
[in a new window]
 


View larger version (14K):
[in this window]
[in a new window]
 
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.

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.


View larger version (11K):
[in this window]
[in a new window]
 
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).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Barnette, M. S., C. D. Manning, W. J. Price, and F. C. Barone. Initial biochemical and functional characterization of cyclic nucleotide phosphodiesterase isozymes in canine colonic smooth muscle. J. Pharmacol. Exp. Ther. 264: 801-812, 1993[Abstract].

2.   Benham, C. D., T. B. Bolton, R. J. Lang, and T. Takewaki. Calcium-activated potassium channels in smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery. J. Physiol. (Lond.) 371: 45-67, 1986[Abstract].

3.   Bolotina, V. M., S. Najibi, J. J. Palacino, P. J. Pagano, and R. A. Cohen. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850-853, 1994[Medline].

4.   Boyle, J. P., M. Tomasic, and M. I. Kotlikoff. Delayed rectifier potassium channels in canine and porcine airway smooth muscle cells. J. Physiol. (Lond.) 447: 329-350, 1992[Abstract].

5.   Carl, A., and K. M. Sanders. Ca2+-activated K channels of canine colonic myocytes. Am. J. Physiol. 257 (Cell Physiol. 26): C470-C480, 1989[Abstract/Free Full Text].

6.   Cole, W. C., and K. M. Sanders. Characterization of macroscopic outward currents of canine colonic myocytes. Am. J. Physiol. 257 (Cell Physiol. 26): C461-C469, 1989[Abstract/Free Full Text].

7.   Conklin, J. L., and C. Du. Guanylate cyclase inhibitors: effect on inhibitory junction potentials in esophageal smooth muscle. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G87-G90, 1992[Abstract/Free Full Text].

8.   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].

9.   Drewett, J. G., and D. L. Garbers. The family of guanylyl cyclase receptors and their ligands. Endocr. Rev. 15: 135-162, 1994[Medline].

10.   Du, C., J. Murray, J. N. Bates, and J. L. Conklin. Nitric oxide: mediator of NANC hyperpolarization of opossum esophageal smooth muscle. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G1012-G1016, 1991[Abstract/Free Full Text].

11.   Farrugia, G., J. L. Rae, and J. H. Szurszewski. Characterization of an outward potassium current in canine jejunal circular smooth muscle and its activation by fenamates. J. Physiol. (Lond.) 468: 297-310, 1993[Abstract].

12.   Giangiacomo, K. M., M. L. Garcia, and O. B. McManus. Mechanism of iberiotoxin block of the large-conductance calcium-activated potassium channel from bovine aortic smooth muscle. Biochemistry 31: 6719-6727, 1992[Medline].

13.   Gimenez-Gallego, G., M. A. Navia, J. P. Reuben, G. M. Katz, G. J. Kaczorowski, and M. L. Garcia. Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels. Proc. Natl. Acad. Sci. USA 85: 3329-3333, 1988[Abstract].

14.   Gold, G. H., and T. Nakamura. Cyclic nucleotide-gated conductances: a new class of ion channels mediates visual and olfactory transduction. Trends Pharmacol. Sci. 8: 312-316, 1987.

15.   Goldman, D. E. Potential impedance and rectification in membranes. J. Gen. Physiol. 27: 37-60, 1943[Free Full Text].

16.   Hamill, O. P., A. Marty, E. Neher, S. Sakmann, and F. J. Sigworth. Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391: 85-100, 1981[Medline].

17.   Hodgkin, A. L., and B. Katz. The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. (Lond.) 108: 37-77, 1949.

18.   Ignarro, L. J., and P. J. Kadowitz. The pharmacological and physiological role of cyclic GMP in vascular smooth muscle relaxation. Annu. Rev. Pharmacol. Toxicol. 25: 171-191, 1985[Medline].

19.   Koh, S. D., J. D. Campbell, A. Carl, and K. M. Sanders. Nitric oxide activates multiple potassium channels in canine colonic smooth muscle. J. Physiol. (Lond.) 489: 735-743, 1995[Abstract].

20.   Miller, C., E. Moczydlouski, R. Latorre, and M. Phillips. Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle. Nature 313: 316-318, 1985[Medline].

21.   Murray, J. A., E. F. Shibata, T. L. Buresh, H. Picken, B. W. O'Meara, and J. L. Conklin. Nitric oxide modulates a calcium-activated potassium current in muscle cells from opossum esophagus. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G606-G612, 1995[Abstract/Free Full Text].

22.   Palmer, R. M. J., A. G. Ferrige, and S. Moncada. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524-526, 1987[Medline].

23.   Perez, G. J., L. Toro, S. D. Erulkar, and E. Stefani. Characterization of large-conductance, calcium-activated potassium channels from human myometrium. Am. J. Obstet. Gynecol. 168: 652-660, 1993[Medline].

24.   Rapoport, R. M., and F. Murad. Effects of ethacrynic acid and cystamine on sodium nitroprusside-induced relaxation, cyclic GMP levels and guanylate cyclase activity in rat aorta. Gen. Pharmacol. 19: 61-65, 1988[Medline].

25.   Rich, A., J. L. Kenyon, J. R. Hume, K. Overturf, B. Horowitz, and K. M. Sanders. Dihydropyridine-sensitive calcium channels expressed in canine colonic smooth muscle cells. Am. J. Physiol. 264 (Cell Physiol. 33): C745-C754, 1993[Abstract/Free Full Text].

26.   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].

27.   Shuttleworth, C. W., C. Xue, S. M. Ward, J. de Vente, and K. M. Sanders. Immunohistochemical localization of 3',5'-cyclic guanosine monophosphate in the canine proximal colon: responses to nitric oxide and electrical stimulation of enteric inhibitory neurons. Neuroscience 56: 513-522, 1993[Medline].

28.   Sims, S. M., B. T. Lussier, and J. Kraicer. Somatostatin activates an inwardly rectifying K+ conductance in freshly dispersed rat somatotrophs. J. Physiol. (Lond.) 441: 615-637, 1991[Abstract].

29.   Singer, J. J., and J. V. Walsh. Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique. Pflügers Arch. 408: 98-111, 1987[Medline].

30.   Stark, M. E., A. J. Bauer, and J. H. Szurszewski. Effect of nitric oxide on circular muscle of the canine small intestine. J. Physiol. (Lond.) 444: 743-761, 1991[Abstract].

31.   Stark, M. E., and J. H. Szurszewski. Role of nitric oxide in gastrointestinal and hepatic function and disease. Gastroenterology 103: 1928-1949, 1992[Medline].

32.   Stockbridge, L. L., A. S. French, and S. F. Man. Subconductance states in calcium-activated potassium channels from canine airway smooth muscle. Biochim. Biophys. Acta 1064: 212-218, 1991[Medline].

33.   Stockbridge, N., H. Zhang, and B. Weir. Potassium currents of rat basilar artery smooth muscle cells. Pflügers Arch. 421: 37-42, 1992[Medline].

34.   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].

35.   Vogalis, F., and K. M. Sanders. Characterization of ionic currents of circular smooth muscle cells of the canine pyloric sphincter. J. Physiol. (Lond.) 436: 75-92, 1991[Abstract].

36.   Wade, G. R., and S. M. Sims. Muscarinic stimulating of tracheal smooth muscle cells activates large-conductance Ca2+-dependent K+ channel. Am. J. Physiol. 265 (Cell Physiol. 34): C658-C665, 1993[Abstract/Free Full Text].

37.   Waldman, S. A., and F. Murad. Cyclic GMP synthesis and function. Pharmacol. Rev. 39: 163-196, 1987[Medline].

38.   Ward, S. M., H. H. Dalziel, M. E. Bradley, I. L. O. Buxton, K. Keef, D. P. Westfall, and K. M. Sanders. Involvement of cyclic GMP in non-adrenergic, non-cholinergic inhibitory neurotransmission in dog proximal colon. Br. J. Pharmacol. 107: 1075-1082, 1992[Abstract].


AJP Gastroint Liver Physiol 274(5):G848-G856
0193-1857/98 $5.00 Copyright © 1998 the American Physiological Society