Introduction

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The canine lower esophageal sphincter (LES) was recently shown to have muscle cells with a membrane-bound and spontaneously active constitutive nitric-oxide synthase (cNOS; Salapatek et al., 1998a). When NOS activity was blocked by N-nitro-L-arginine (L-NOARG) after enteric nerve activities were inhibited by tetrodotoxin (TTX) and -conotoxin (GVIA) [-CTX(GVIA)], tone persistently increased. However, when entrance of Ca²⁺ through L-type Ca²⁺ channels was inhibited, tone was lost and the ability of L-NOARG to increase tone also disappeared. Studies of single muscle cells in whole-cell patch-clamp demonstrated large outward K⁺ currents on depolarization when intracellular Ca²⁺ concentration ([Ca²⁺]_i) was 200 nM. These were reduced ~80% by 100 nM iberiotoxin (Ibtx). L-NOARG also inhibited these outward currents by ~80%, and this effect was obtunded when Ibtx was present (Salapatek et al., 1998b). These outward currents were dependent on the level of [Ca²⁺]_i determined by EGTA buffers in the pipette; currents were minimal at 8 nM and increased with pipette [Ca²⁺]_i, reaching a maximum at ~200 nM and with an EC₅₀ value of 108 nM. They were also inhibited when L-type Ca²⁺ channels were blocked. An NO donor like sodium nitroprusside (SNP) had no effect on outward currents when pipette [Ca²⁺]_i was 200 nM. SNP restored them fully when they were reduced by L-NOARG or by 8 nM [Ca²⁺]_i but not at all when they were inhibited by Ibtx. In current clamp mode, these cells had membrane potentials ~45 mV, and cells were depolarized by L-NOARG or Ibtx and restored in membrane potential by NO donors except when Ibtx was used. From these studies, we concluded that the cNOS in canine LES was spontaneously active, depending on Ca²⁺ entry through L-type Ca²⁺ channels, to produce NO to activate large conductance Ca²⁺-dependent K⁺ channels (BK_Ca channels), hyperpolarize the membrane, and limit Ca²⁺ entry and tone.

The aim of this study was to evaluate the effects of K⁺ channel blockade on relaxation of tone when NO was derived from the muscle, when it was provided exogenously, and when it was derived from enteric nerves. Previous studies have shown that inhibition from enteric nerve stimulation in canine LES depends on hyperpolarization and relaxation mediated by NO (Jury et al., 1992). Our initial results led us to examine the roles of other K⁺ channels besides BK_Ca channels and of Cl channels in mediating neural relaxation as well as relaxation from exogenous NO donors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Mongrel dogs, chosen regardless of gender, were euthanized with an overdose of sodium pentobarbital (100 mg/kg) in accordance with a protocol approved by the McMaster Animal Ethics Committee and the guidelines of the Canadian Council for Animal Care. The gastroesophageal region was then carefully removed from the dog and placed into a cold (4°C) Krebs-Ringer solution composed of 115.0 mM NaCl, 4.6 mM KCl, 22.0 mM NaH₂PO₄, 2.5 mM CaCl₂, and 11.0 mM glucose. The Krebs-Ringer solution was also equilibrated with 5% CO₂, 95% O₂. The gastroesophageal junction was then opened on the gastric greater curvature side, and the mucosa was removed by sharp dissection. This revealed the LES as a thickened ring of muscle composed of clasp fibers with oblique gastric sling fibers on either side. The LES used for experimentation was taken only from the clasp region of the LES.

In Vitro Studies

Recording of Mechanical Activity. After the muscle bundles were revealed and identified in the LES region, circular muscle strips ~40 mm × 2 mm were dissected out of the clasp fiber region of the LES. The muscle strips were then securely tied with silk ligature after being cut into 10 mm × 2 mm strips. Muscle strips were then hung in 5-ml organ baths, bathed in Krebs-Ringer solution (a Krebs-Ringer salt solution of composition given earlier) at a temperature of 37°C, and bubbled with 95% O₂, 5% CO₂. The strips were orientated vertically in the baths with the bottom end affixed to an electrode holder by silk ligature, and the top end was passed through a pair of concentric platinum electrodes and then affixed to a longer silk thread and ligature to a force displacement transducer (Grass FTOC3). Two grams of tension was applied initially to each strip. Strips were allowed to equilibrate for a period of 1 h. During the equilibration period, the muscle strips contracted and spontaneously developed tone. Active tension was taken to be the difference between the observed tension and the minimum tension that was obtained at the conclusion of each experiment in Ca²⁺-free physiological salt solution (PSS; made by adding no Ca²⁺ but 1 mM EGTA). The changes in tension of the muscle strips were displayed on an eight-channel Beckman R611 Dynograph. A Grass S88 stimulator set at 40 V, 5 pps, 10 s (unless otherwise noted) with 0.1-, 0.2-, and 0.3-ms pulse durations applied sequentially for 10 s generated the electrical field stimulations (EFSs). It was previously shown that these stimulation parameters produce TTX-sensitive relaxations (Allescher et al., 1988; Salapatek et al., 1998a), and these studies reconfirmed this finding.

Experimental Protocols and Drugs Used

The muscle strips were left to equilibrate for 1 h to allow spontaneous tone to develop. If this did not occur, strips were discarded. EFSs were applied to all the strips at the above settings and produced relaxations.

Studies without Nerve Function. In studies with nerve activity blocked, 10⁶ M TTX and 100 nM -CTX(GVIA) were then added to the Krebs' solution throughout the remainder of the study. In some experiments, -CTX(GVIA) alone was added to determine whether the source of NO for relaxation was entirely abolished by this agent. Thirty minutes after the toxins were added and EFSs were retested to ensure that nerves were blocked, either L-NOARG, an NOS inhibitor (10⁴ M), Ibtx (100 nM), tetraethyl ammonium (TEA; 20 mM), or 4-aminopyridine (4-AP; 5 mM), or combinations of these, were applied. If L-NOARG was applied to some strips, other strips received other agents. Then after L-NOARG had produced a stable increase for 15 min, Ibtx, TEA, or 4-AP was added to determine whether additional tone increase occurred. In strips that received K⁺ channel blockers first, after stable tone increases for 15 min, L-NOARG was added to determine whether further tone increase occurred. In every experiment, one or two strips were left with no additions or only L-NOARG added. Then, 10⁴ M SNP or 3-morpholino-sydnonimine (Sin-1) was added to all strips, and the extent of relaxation over 15 min was examined. Finally, all tissues were exposed to Ca²⁺-free Krebs' solution with 100 µM EGTA to determine basal tone. In later studies, only SNP was used.

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Studies with Nerves Active. These studies were carried out without adding TTX or -CTX(GVIA) but following a similar design. However, as K⁺ channels are present on nerves, we evaluated the possibility that effects from these agents on tone were a consequence of actions on nerves, as described later.

Role of Cl Channels in Relaxation Responses to EFS. The same general approach was used but EFS was tested before and after increasing concentrations of Cl channel blockers. We used niflumic acid (NFA; 10-100 µM) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 500-1000 µM). Because each produced concentration-dependent loss of tone, relaxations to EFS were subsequently examined in the presence of carbachol- or TEA-induced tonic and phasic activity as well as in terms of the level of tone at the nadir of relaxation. We also examined the effect of replacing all of the NaCl with sodium isethionate, leaving 9.6 mM Cl in the medium. This was intended to eliminate any inward electrochemical gradient for Cl, which is estimated to have an E_Cl value of ~35 mV, with a membrane potential of ~45 mV. In several experiments in which tested agents affected tone, to save space, we report only the degree of relaxation achieved during EFS at 0.3-ms duration pulses, called residual tone, and expressed it in relation to initial tone.

Patch-Clamp Studies

Cell Isolation. Pieces of canine LES were dissected as previously described (Salapatek et al., 1998a). Circular smooth muscle strips were cut into 1- to 2-mm² pieces and placed in a dissociation solution containing 0.25 mM EDTA, 125 mM NaCl, 4.8 mM KCl, 10 mM glucose, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES for 30 min. An enzyme solution containing papain (130 mg/ml), ()-1,4-dithio-L-threitol (15.4 mg/ml), BSA (100 mg/ml), and one of the Sigma collagenase blends L, H, or F (130 mg/ml), was added to the tissue pieces incubated at 37°C for 30 to 60 min. After incubation, the enzyme solution was decanted off, and the tissue pieces were rinsed in enzyme-free dissociation solution. Single cells were gently mechanically agitated with siliconized Pasteur pipettes to disperse tissue and isolate single smooth muscle cells. Cells used in this study were patch-clamped at room temperature (22-24°C) usually within 8 h of isolation.

Patch-Clamp. The cell suspension was placed in a glass-bottomed dish. Within 30 min, cells adhered to the dish. The cells were then washed by perfusion with Ca²⁺-containing external solution (140.0 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl₂, 1.0 mM MgCl₂, 10.0 mM HEPES, 5.5 mM glucose, pH adjusted to 7.35 with NaOH). Patch electrodes were made using borosilicate glass capillary tubes using a Flaming Brown micropipette puller (Sutter Instruments Inc., Novato, CA). Pipettes were polished using a microforge (Narishige MF-83) to resistance of 3 to 5 M. The pipette solution contained 2.5 mM CaCl₂, 140 mM KCl, 1 mM MgCl₂, 10 mM HEPES, and 4 mM Na-ATP and EGTA to obtain an [Ca²⁺]_i of 200 nM, using MAX Chelator software.

Drugs and Chemicals

Unless otherwise stated, drugs in this study were purchased from Sigma Chemical Co. (St. Louis, MO), were of the highest quality available, and were dissolved in PSS on the day of the experiment. L-NOARG was first dissolved in 0.01 N HCl to make a 10² M solution, which was diluted 100-fold for use. A control using HCl had no action on the LES by itself. Ibtx was donated by Dr. I. Rodger (Merck Frosst Canada) and was dissolved in double distilled water; small aliquots were stored at 22°C until needed. -CTX(GVIA), TTX, TEA, 4-AP, L-arginine, atropine, and SNP were made up on each experimental day as stock solutions 100× more concentrated than needed for muscle bath studies. SNP was kept in a bottle wrapped in aluminum foil to avoid photolysis. NFA was dissolved in ethanol at 10¹ M and then diluted in PSS to make 10 to 100 µM (10⁵ to 10⁴ M). DIDS was dissolved in DMSO at 10¹ M and diluted to 10⁴ to 10³ M for application to the strips. At the highest concentrations of DIDS, the DMSO had relaxant effects as described in Results.

Analysis

Tone at various stages of the experiments, relaxations, and/or area under the tension trace was measured. Tone changes were evaluated in initial experiments, when block of K⁺ channels compared with block of NO synthesis was compared, in terms of the percentage of change from the basal, initial tension of 2 g. In later experiments when inhibition of nerve-stimulated relaxations was studied, these changes as well as relaxations were evaluated in terms of the percentage of the steady-state, initial, active tone achieved by each muscle strip. Effects on relaxations to SNP were evaluated by comparing area under the tension traces for 0 to 5 and/or 5 to 10 min after SNP to a similar area under the steady-state initial tone. The area under the curve (AUC) was measured using a computerized microplanimeter (Laboratory Computer Systems, Cambridge, MA). Data were expressed as mean ± S.E., and the mean values were expressed as a percentage of the baseline active tension or the rectangular area defined by the LES tone before drug addition and a set period of time (5 min) to standardize the data. Active tension was defined as the tension above that achieved after exposure to Ca²⁺-free medium with 1 mM EGTA. This value was usually the same as the initial applied tension. The number of experiments, which also represented the number of animals, was indicated by n. One-way ANOVA or, when appropriate, a repeated measures ANOVA was used to analyze the data. Either Tukey-Kramer multiple comparisons test or Bonferroni's correction was used to determine the statistical significance of differences between means. P values less then .05 were considered significant (*P < .05, **P < .01, ***P < .001).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

What K⁺ Channels Mediate Effects of Myogenic NO on LES Tone? Ibtx or L-NOARG was each previously shown (Salapatek et al., 1998b) to depolarize cells by 8 to 10 mV and to abolish ~80% of the outward current carried by K⁺ ions when cells were maximally depolarized and had [Ca²⁺]_i of 200 nM. We expected that its effects to increase tone would be equivalent to those of L-NOARG in the absence of nerve activity. As illustrated in Fig. 1 (top) and summarized in Fig. 1 (bottom), it increased tone, but subsequent L-NOARG (10⁴ M) increased it further. When given in reverse order, Ibtx, after L-NOARG, had no additional effect. Ibtx (10⁷ M) only slightly reduced the relaxation to subsequent SNP (10⁴ M) whether given alone or after L-NOARG, which by itself had no effect (Fig. 2). TEA at 20 mM concentrations acts nonselectively on many K_V channels as well as on BK_Ca channels. It increased tone more than Ibtx (Fig. 3, top) and occluded any further tone increase to L-NOARG or to either 4-AP (5 mM) or Ibtx (Fig. 3, bottom). TEA alone or with additional K⁺ channels blockers reduced relaxation to SNP or Sin-1 more than did Ibtx. However, the inhibition was incomplete, amounting to 50 to 55% of the active tone present in the period from 5 to 10 min after SNP (Fig. 2). These results suggested that: 1) additional K⁺ channels blocked by high TEA concentrations, besides Ibtx-sensitive BK_Ca channels, mediated the inhibition of tone by myogenic NO, and 2) these channels and channels or mechanisms, unlikely to be openings of K⁺ channels because they were not susceptible to high concentration of both TEA and 4-AP, mediated relaxation to exogenous NO. To assess whether unusual K⁺ channels, resistant to TEA and 4-AP, were present, we evaluated the effects of these agents on outward K⁺ currents.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Comparison of LES tone increases from Ibtx and L-NOARG when nerves were inactive. Top, portions of a representative experiment showing that Ibtx raised tone when nerves were blocked and that subsequent L-NOARG (104 M) increased tone further. Basal tone represents the tension initially set (2 g) before tone recovery and the addition of TTX and -CTX(GVIA) and before any K+ channel blocker. After the addition of Ibtx and L-NOARG, SNP was added and left for 15 min; then, the medium was replaced with Ca2+-free Krebs' solution containing 1 mM EGTA for 10 min The removal of all Ca2+ restored the passive basal tone. The difference between basal tone and tone after SNP was measured as AUC (usually 5-10 min after addition of SNP) to indicate the degree to which relaxation to SNP had been blocked. Bottom, experiments in which either L-NOARG or Ibtx was given first. When L-NOARG was given first, subsequent Ibtx had no effect. However, when Ibtx was given first, the tone increase was less than when L-NOARG was given first (P < .01), and subsequent L-NOARG increased it to the same level as when L-NOARG was given first (n = 6).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Comparison of abilities of Ibtx and KV channel block to decrease relaxant effects of NO donors. Top, representative tracing of experiment in which TEA was added before other agents and occluded any additional tone increase when 4-AP, Ibtx, and L-NOARG were added. Then, SNP was added, but now relaxation was incomplete. Bottom, residual tone after SNP or SIN 1 as AUC for a 5-min interval. AUC was determined just before and after each NO donor had produced maximal relaxation. This area was not different from zero AUC when L-NOARG alone was used (not shown). Note that Ibtx increased this area to ~24%, whereas the combined blockers increased significantly more by 50 to 57% (n = 6).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Comparison of abilities of Ibtx and KV channel block to increase LES tone, relative to tone increase by L-NOARG. Top, after 20 mM TEA, neither 5 mM 4-AP, Ibtx, nor L-NOARG caused a further tone increase. Bottom, after Ibtx, TEA increased tone significantly but no further increase occurred with 4-AP or L-NOARG (n = 6).

Effects of K⁺ Channel Blockade on Outward Currents. In whole-cell recordings (Fig. 4), TEA given first and 4-AP given second at the concentrations used on tissues decreased outward currents, and the combination of the two inhibitors reduced them even more. The results were the same regardless of whether the cells were tested by adding TEA and 4-AP in sequence or by study after both were present. In three experiments, 10⁷ M apamin was added after the other agents and produced no additional inhibition of outward currents. Small residual currents, like those present when Cs⁺ was substituted for K⁺ (Salapatek et al., 1998b), probably were carried by other ions such as Cl. These results support the suggestion that the combination of high concentrations of TEA and 4-AP blocks all outward currents through K⁺ channels, and that other channels or mechanisms mediated relaxations to NO donors in part.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of TEA and 4-AP on membrane currents in isolated LES cells. Top, changes in current-voltage relationships for outward K+ currents after 20 mM TEA and after TEA and 5 mM 4-AP. Also included and illustrated () are five cells patched after both TEA and 4-AP were present; the results were similar to those in which recordings were made sequentially. Access resistances were 10.9 ± 1.4 M, and capacitances were 55.5 ± 12 pF. In three experiments (not shown), apamin (107 M) was subsequently added to no effect (n = 5). Bottom, representative set of current traces when TEA and 4-AP were added sequentially.

What Ion Channels Mediate the Relaxant Effects of NO from Enteric Nerves? When these studies were repeated in the absence of 10⁶ M TTX or 10⁷ M -CTX(GVIA), we obtained unexpected results. TEA still raised tone as much as when nerves were blocked by TTX (Fig. 5, top), but relaxations to EFS were not inhibited (Fig. 5, middle). Although the amplitudes of relaxation were increased because of the increased tone, residual tone at the nadir of relaxation was unchanged. However, relaxations to SNP were less when nerves were unblocked than when TTX was present (Fig. 5, bottom). This suggested that TEA might release an excitatory mediator when enteric nerves were functioning.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effects of 20 mM TEA on LES tone with or without nerve block and on relaxations to EFS and SNP. Top, comparison in tissues in the same experiment, the increase in tone relative to initial tone (stable level of tone achieved after recovery) by 20 mM TEA with or without inhibition of nerve activity by TTX. There was no significant difference. Middle, the residual tone (tone level above tone in zero Ca2+) at the nadir of the relaxation to 0.3-ms EFS was unchanged by TEA treatment. Bottom, effect of TEA on areas under the curve (AUCs) (tone in excess of that in zero Ca2+) for the first and second 5-min intervals after the addition of SNP. Results were compared with tone during the 5-min interval before SNP. Note that when TTX was present, the degree of relaxation was significantly greater (*P < .05, **P < .01) (i.e., residual tone was less) (n = 4).

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View larger version (47K): [in this window] [in a new window]   Fig. 6.   Effect of atropine on responses of LES tone to TEA and on ability of TEA to decrease relaxation to SNP. Top, in the absence of TTX, 107 M atropine had no significant effect on the increase in tone from 20 mM TEA. Bottom, pretreatment with atropine did not affect the AUCs (measured from the zero Ca2+ baseline) for the first 5-min interval and the 5-min interval 10 min after the addition of SNP. There were no significant differences (n = 3).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Effects of 5 mM 4-AP on LES tone and relaxation to EFS of L-NOARG on relaxation to 4-AP. Top, tone decreased when nerves were active on the addition of 4-AP. Columns labeled NT (no treatment) refer to measurement at two successive times after EFS and before any treatment showing the stability of tone (n = 3), Bottom left, 104 M L-NOARG nearly abolished (not significantly different from zero) relaxation to EFS at 0.3 ms. After 4-AP, no relaxation could be detected (n = 9). Top right, after L-NOARG, 4-AP no longer relaxed the LES (n = 9). Bottom right, effects of 4-AP with or without TTX compared with the stable initial tone. TTX reversed the effect of 4-AP from tone decrease to tone increase (n = 3).

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effects of L-NOARG on relaxations to EFS and to 4-AP in LES. Top, relaxations to EFS at 0.3-ms duration after 1 mM L-arginine (L-ARG), which alone had no effect (not shown) but prevented a significant inhibition of relaxation after L-NOARG. After the addition of 4-AP, relaxations to EFS were still not significantly changed (n = 9). Bottom, companion study in strips from the same experiments showing that L-ARG did not prevent L-NOARG from abolishing, often reversing, any relaxation to 4-AP (n = 4).

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Does Closure of Cl Channels Mediate Relaxation from EFS? In opossum body circular muscle and other gut muscles, it has been reported that Cl channel closures mediate NFA-sensitive inhibitory junction potentials and relaxations from EFS (Christ et al., 1991a,b; Zhang et al., 1998). We tested whether this was the case for our tissues, using NFA. NFA, in concentrations from 10 to 100 µM, concentration dependently reduced tone and 100 µM nearly abolished it (Table 3), as reported for Cl channel blockers in arteries (Nelson et al., 1997). To determine whether this also affected EFS-induced relaxations we evaluated whether EFS still reduced residual tone or restored tone (usually accompanied by phasic activity) produced by adding 10⁶ M carbachol or 20 mM TEA to the bath. When tone was present or restored by carbachol (Table 3), EFS, applied during a plateau of phasic contraction, still caused relaxations and/or inhibition of phasic activity down to the same residual tone as control. After 100 µM NFA, TEA at 20 mM restored tone and EFS still relaxed any tone present. Moreover, NFA had no ability to reduce relaxations to SNP. We concluded that NFA was ineffective to inhibit nerve-mediated relaxations of LES or those from NO donors. We tested an additional putative Cl channel blocker, DIDS.

View this table: [in this window] [in a new window]   TABLE 3 Effects of putative Cl channel blockers on tone and EFS (0.3 ms) responses

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	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

NO synthesized in LES appears to modulate tone by opening of Ibtx-sensitive BK_Ca channels (Salapatek et al., 1998a,b). Here, we show that K_V channels insensitive to Ibtx but sensitive to 20 mM TEA are also involved. However, NO from nerves still relaxed when all K⁺ channels are blocked. No role of Cl channels was found. NO donors appeared to act partly by opening K⁺ channels, but partly by other mechanisms not involving Cl channels. These statements depend on the reliability of the pharmacological tools used.

With nerves inactive, we compared the increase of tone when NO from myogenic NOS was inhibited to the tone increases when various K⁺ channels were blocked. We assumed that occlusion of any increase in tone from L-NOARG after K channel blockers raised tone, provided insight into whether the channels blocked were those affected by myogenic NO. We used the partial reduction of relaxation to SNP on blocking K⁺ channels to indicate their role in its relaxing mechanism.

To block K_V channels as well as BK_Ca channels, we used TEA and 4-AP at high concentrations. High concentrations of TEA block BK_Ca channels and most voltage-dependent K_V channels; adding 4-AP blocks voltage-dependent channels insensitive to TEA, except inward rectifier channels. The effectiveness of this combination to block all other K channels in a variety of smooth muscle is well established (Nelson and Quayle, 1995; Vogalis et al., 1996; Horowitz al., 1999; Hurley et al., 1999). Moreover, we showed that in single LES muscle cells with 200 nM pipette Ca²⁺, the combination of 20 mM TEA and 5 mM 4-AP nearly abolished outward currents, and subsequent apamin had no additional effect. Thus, nearly all K⁺ channels were likely inactive in the presence of these two antagonists. Studies of tone increase when nerves were inactive showed that 20 mM TEA alone produced contractions equivalent to or greater than L-NOARG or any other K⁺ channel antagonist, suggesting that channels blocked by TEA included those opened by myogenic NO. We are unaware of any TEA action, besides block of K⁺ channels, likely to increase tone in the absence of functioning nerves.

Ibtx blocks BK_Ca channels selectively (Horowitz et al., 1999; Hurley et al., 1999). It and L-NOARG each obliterated the same 81% of the outward current in LES cells at depolarized conditions when pipette Ca²⁺ was 1000 nM (Salapatek et al., 1998b). Ibtx increased tone less than L-NOARG or TEA, suggesting that other K_V channels opened in response to myogenic NO. In canine colon myocytes, Koh et al. (1995) found that two K⁺ channels besides BK_Ca opened in response to NO donors, consistent with our interpretation. We detected no difference previously in the reduction in outward currents between L-NOARG and Ibtx (Salapatek et al., 1998b), presumably because we examined currents under depolarized and high [Ca²⁺]_pipette conditions when BK_Ca currents are very large and after a long depolarizing ramp, allowing inactivation of other K_V currents. In this study, using a 200 nM pipette [Ca²⁺] and depolarizing steps instead of ramps, 20 mM TEA inhibited less than 70% of the outward currents. This [Ca²⁺]_pipette concentration more nearly approaches the physiological level present in a sphincter with active tone than the 1000 nM used previously (Salapatek et al., 1998b). Thus, myogenic NO appears to act on TEA-sensitive K⁺ channels, including BK_Ca channels, but not on additional 4-AP-sensitive channels.

Relaxations to NO donors, SNP or Sin 1, were reduced slightly but significantly by Ibtx and more (~50%) by TEA. 4-AP had little additional effect on relaxations from SNP, again suggesting that channels resistant to TEA but sensitive to 4-AP are not involved in NO effects. NO is released from SNP by membrane-located enzymes inside cells (Kowaluk et al., 1992; Ferrero et al., 1999). NO released near the membranes of LES should affect the same muscle K⁺ channels as when NO is released by local neural NOS. This prediction was confirmed in that block of K⁺ channels by TEA, alone or with 4-AP, reduced responses to SNP. However, these agents were incompletely effective to prevent SNP relaxation, as also reported for carotid artery (Plane et al., 1998). Therefore, NO donors had sites of action involving K⁺ channels and sites not involving K⁺ channels, including sites not affected by NO synthesized in the LES cells.

Relaxation from nerve stimulation was not reduced by TEA. This was surprising because NO increases outward currents in LES cells (Salapatek et al., 1998b; Jury and Daniel, 1999). The amplitudes of relaxation increased because TEA increased tone, but residual tone during EFS was unchanged. TEA affected relaxations from exogenous NO donors similarly as in the absence of nerve function. This implies that NO released on EFS does not reach LES muscle, does not affect the K⁺ channels activated by intramuscular NO and by NO from donors, or acts by another mechanism that bypasses a requirement for function of K⁺ channels. NO diffuses rapidly and freely, easily passing through cell membranes, but it is rapidly and spontaneously oxidized (Moncada et al., 1991).

After the addition of TEA or 4-AP relaxations were still abolished by L-NOARG (i.e., were still dependent on NO release). However, K⁺ channel blockers affected enteric nerve function. Atropine reduced the effect of TEA to decrease relaxations to SNP when TTX was absent. Thus acetylcholine may have been released by TEA. In contrast to TEA, which caused similar contractions with or without nerve activity, 4-AP caused contractions when nerves were inactive and relaxations when nerves were active. These relaxations were blocked by L-NOARG, suggesting that inhibition of 4-AP sensitive K⁺ channels in nerves activated NO release. However, in contrast to relaxations to EFS, 4-AP-induced relaxations did not depend on functioning N-Ca²⁺ channels. Block of 4-AP relaxation by L-NOARG was not reversed or protected by L-arginine, even though relaxations to EFS were restored or protected. Why L-arginine had this selective effect is unknown. 4-AP-induced relaxations were increased after atropine, suggesting that it releases acetylcholine as well as NO.

NO from nerves may not require K⁺ channel opening to initiate relaxation because it may act on interstitial cells of Cajal (ICC) instead of muscle. Structural arrangements in canine LES (Berezin et al., 1987; Allescher et al., 1988; Daniel and Berezin, 1992) show that NO from nerve endings might act directly on ICC before reaching muscle. ICC might respond to NO by closing Cl channels (Christ et al., 1991a,b), hyperpolarizing them and LES muscle cells passively through gap junction contacts. We tested this hypothesis and a general role for Cl channel closure by adding NFA, a putative blocker of Ca²⁺-activated Cl channels or DIDS, a blocker of these as well as other Cl channels and HCO₃-Cl exchange (Clapp et al., 1996; Salter and Kozlowski, 1996). Cl channels closure might inhibit basal Cl efflux that contributes to the low (40 to 45 mV) membrane potentials of LES cells (Jury et al., 1992; Salapatek et al., 1998b), hyperpolarizing and relaxing them.

As expected if it hyperpolarizes cells, NFA dose-dependently inhibited tone in LES, secondarily reducing amplitudes of relaxations to EFS. However, the extent of relaxation achieved by EFS was not affected, and when the tone was restored, accompanied by phasic activity, after the addition of carbachol or TEA, relaxations were also restored. NFA may not block only Ca²⁺-activated Cl channels. A recent report (Kato et al., 1999) found that in pulmonary arteries, NFA acted to inhibit contractions to endothelin-1 by a mechanism independent of Cl channel blockade. Ottolia and Toro (1994) and Greenwood and Large (1995) reported that NFA activated BK_Ca channels. This could also explain the relaxation of tone. Because 20 mM TEA added in the presence of 100 µM NFA restored tone, K⁺ channel opening is likely to participate in the relaxation to NFA. Moreover, NFA alone failed to significantly reduce relaxation to SNP. This result suggests either that Cl channels play no essential role in relaxations induced by EFS or by NO donors in canine LES or that NFA is not a Cl channel blocker in this tissue.

DIDS, which unlike NFA had no effect on endothelin-1 contractions of pulmonary artery (Kato et al., 1999), partially relaxed LES tone, mainly caused by the DMSO present with it. DIDS alone had no effect on EFS relaxations, assessed in terms of the residual tone at the nadir of the response or on relaxations to SNP. However, together with TEA (20 mM), DIDS nearly abolished relaxation to EFS. This suggested the possibility that a combination of K⁺ (opened) and Cl (closed) channels are affected by NO released from nerves and that either action alone is capable of relaxing the LES.

However, when we substituted sodium isethionate for NaCl, thereby lowering [Cl]_e to 9.6 mM, relaxations were not inhibited after tone was restored, even when this was achieved with TEA. If DIDS were acting through a Cl channel closure mechanism, the reversal of the Cl gradient should have the same effect, and combined with TEA, relaxation to EFS should have been abolished. There was a significant reduction in the degree of relaxation, but clear relaxation still occurred. Only when DIDS was added after TEA (not after carbachol) were relaxations to EFS abolished. It is possible that reversal of the Cl gradient affects other cell functions such as the ability of the sarcoplasmic reticulum to pump Ca²⁺ (Daniel et al., 1992). Therefore, the mechanism or mechanisms of the combination of TEA and DIDS to inhibit relaxations by nerve-mediated release of NO remains unknown. The fact that this combination failed to completely block the relaxation response to SNP shows that TEA- and DID-resistant, as well as TEA-sensitive, multiple mechanisms are involved in relaxations to NO donors.

A dual action of neural NO to relax LES might result from local action on ICC as well as on muscle. Hyperpolarization of ICC would be transmitted to LES by gap junctions (Berezin et al., 1983; Allescher et al., 1988). ICC in this region, as in others, often appear to have NADPH/diaphorase (Wang et al., 1998) and may be capable of generating NO after activation. In mice lacking ICC in the LES (Ward et al., 1998), hyperpolarization and relaxations to EFS and to SNP were attenuated. Therefore, NO from nerves may activate ICC, which somehow amplify that message to hyperpolarize both their membranes and the membranes of cells to which they are coupled.

Direct evidence establishing the presence of Cl channels and whether DIDS or NFA blocks them is needed to clarify our findings. However, our findings show that neurally derived NO has a different, additional site of action from myogenic NO. Because the combination of DIDS and TEA did not completely inhibit relaxations to SNP, it is likely that a further mode of NO relaxing action exists, possibly involving neither K⁺ nor Cl channels, such as a change in the interaction of [Ca²⁺]_i with the contractile apparatus.