Introduction

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Previously we showed that canine LES contains a membrane-bound, constitutive nitric-oxide synthase (Salapatek et al., 1998b,c) that acts to limit development of tone in canine LES. This nitric-oxide synthase, which appears to be neural (Daniel et al., 2001a), uses Ca²⁺ entering through nearby L-type calcium channels for activation and tone regulation by NO formation that activates K⁺ channels, including BK_Ca channels (Daniel et al., 2000). However, the source is not neural as tetrodotoxin alone or with -conotoxin (GVIA) had no effect on tone (Salapatek et al., 1998a,c). Moreover, tetrodotoxin alone but not -conotoxin (GVIA) alone abolished relaxations to electrical field stimulation (Daniel et al., 2000).

Another study showed that contraction in LES was supported by two extracellular sources of Ca²⁺, one of which supported spontaneous tone whereas the other, which supported contraction to carbamyl choline (carbachol), was resistant to removal by extracellular fluid with 0 Ca²⁺ and 100 µM EGTA and appeared to recycle between extracellular binding sites and nearby Ca²⁺ stores (Salapatek et al., 1998a). Similar results have been obtained in airway smooth muscle (Montano et al., 1993, 1996; Bazan-Perking et al., 1998). In these studies, Ca²⁺ from both of these sources entered through L-type calcium channels. We suggested that the Ca²⁺ bound extracellularly was located in membrane caveolae, which also contained neuronal nitric-oxide synthase and L-type calcium channels based on studies in another canine smooth muscle and recently in LES (Darby and Daniel, 2000; Daniel et al., 2001a).

We showed (Daniel et al., 2000) that relaxation by NO released from LES enteric nerves was unaffected by the same potassium channel blocking agents that inhibited NO-mediated muscle relaxation and outward currents. These potassium channel blocking agents, however, partially inhibited relaxations to exogenous NO whether delivered from sodium nitroprusside or 3-morpholinosydnonimine. NO-mediated LES relaxation was also unaffected by chlorine channel blockade. However, a combination of TEA and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid abolished nerve-mediated relaxations. These findings raised the question: are the same second messengers involved in signaling the action of NO when it is derived from nerves, from muscle, or added exogenously?

NO signaling has been studied in many tissues and often involves activation of cytosolic guanylate cyclase with elevation of cGMP, activation of protein kinase G, and subsequent phosphorylation of various membrane proteins, as exemplified in recent references (Ignarro et al., 1999; Chang et al., 2000; Gorodeski, 2000a,b; Janssen et al., 2000; Kwan et al., 2000; Tseng et al., 2000; Yao et al., 2000). However, an increasing number of NO-mediated biological events do not use cGMP or protein kinase G (Ahern et al., 1999; Ignarro et al., 1999; Pinilla et al., 1999; Taglialatela et al., 1999; Garry et al., 2000; Janssen et al., 2000; Liu et al., 2000; Mazzuco et al., 2000; Takeda et al., 2000; Tseng et al., 2000). We aimed to evaluate the roles of guanylate cyclase in the downstream events initiated by NO released from nerves and from muscle.

	Materials and Methods

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Tissue Preparation. Mongrel dogs of either sex were euthanized with an intravenous overdose of pentobarbital sodium (100 mg/kg), according to a protocol approved by the McMaster University Animal Care Committee and following the guidelines of the Canadian Council on Animal Care. The abdomen was opened along the midline, and segments of lower esophagus, ileum, and colon were excised and immediately put into oxygenated Krebs-Ringer solution at 24°C having the following composition: 115.0 mM NaCl₂, 4.6 mM KCl, 1.2 mM MgSO₄, 22.0 mM NaHCO₃, 2.5 mM CaCl₂, and 11.0 mM glucose. The gastroesophageal junction was removed and opened along the greater curvature. After careful removal of the mucosa by fine dissection, the thickened ring of muscle, the LES, was removed. The mucosa was removed by fine dissection, leaving the muscularis externa.

In Vitro Studies. In all cases circular muscle strips were prepared by cutting tissues into multiple 15 × 2 mm strips . These were tied with fine thread at both ends and mounted vertically in 5-ml organ baths, bathed in Krebs-Ringer solution at 37°C, and oxygenated with 95% O ₂ and 5% CO_{2 .} Strips were tied at the bottom to an electrode holder, passed through concentric platinum electrodes, and tied at the top to a force displacement transducer (Grass FT OC3; Grass Instruments, Quincy, MA). Tensions were recorded on Beckman R611 Dynagraphs (Beckman Coulter, Inc., Fullerton, CA). Electrodes were stimulated from a Grass 88 stimulator set at 40 V/cm, 5 pps, and 0.3-ms pulse duration, which gives near maximal relaxation of LES by activation of enteric nerves.

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Patch-Clamp Techniques. The LES was dissected as described above and strips were cut into 1- to 2-mm² square pieces and placed in the dissociation solution.

Cell Isolation. Cells were dissociated in 0.25 mM EDTA, 125 mM NaCl, 4.8 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM glucose for 30 min. An enzyme solution containing papain (130 mg · ml¹), 1,4-dithio-L-threitol, (15.4 mg · ml¹), bovine serum albumin (100 mg · ml¹), and Sigma collagenase blend H (occasionally F) was added to the tissue pieces 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 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 Methodology. Cells from the suspension were 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 that contained 140 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 5.5 mM glucose, pH adjusted to 7.35 with NaOH. Patch electrodes were made using borosilicate glass capillary tubes and a Flaming Brown micropipette puller (Sutter Instrument Co., Novato, CA). After polishing with a microforge (Narishige MF-830; W. Nuhsbaum, Inc., McHenry, IL) and filling, pipettes had resistances of 2 to 5 M. High Ca²⁺ pipette solution contained 2.5 mM CaCl₂, 140 mM KCl, 1 mM MgCl₂, 10 mM HEPES, 4 mM sodium-ATP, and 0.3 mM EGTA to obtain free Ca²⁺ of 8 µM. CaCl₂, KCl, and EGTA levels were adjusted to obtain 200 nM free Ca²⁺ levels as calculated using MAX Chelator software (version 6.72) by Bers et al. (1994).

Drugs Used. ODQ was obtained from Sigma-Aldrich Canada (Oakville, ON, Canada). All other drugs were purchased from Sigma-Aldrich (St. Louis, MO).

Data Analysis. Tone and relaxation nadirs were measured in LES in each strip. Each was compared with the control values as 100% and to the observed initial nadir of relaxation. Changes with concentrations of inhibitor were evaluated using Dunnett's multiple comparisons test unless only one concentration was used. Then paired comparisons were made. All statistical tests were carried out using Prism 3 software (Intersil Corp., Irvine, CA). The n values in tables refer to the number of animals that supplied the tissues.

	Results

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Drug Effects on Active Tone with/without Functioning Nerves. In the absence of nerve blockade with 10⁶ M tetrodotoxin, ODQ (10⁵ M) and TEA (20 mM) both increased active tone, TEA more than ODQ (cf. Figs. 1, 2, and 3, B and C). Tetrodotoxin alone or combined with -conotoxin (GVIA) had no effect on tone (Salapatek et al., 1998b). When tetrodotoxin had blocked responses to electrical field stimulation, TEA, but not ODQ, still increased active tone (Fig. 2 and Table 1), and ODQ after TEA had no additional effect. Moreover, the increase in tone from TEA was greater after tetrodotoxin than before, presumably a result of removal of TEA-induced release of inhibitory nerve mediator.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effects of 105 M ODQ on tone (compared with tone prior to adding ODQ, both expressed relative to original tone of 100%). Note that ODQ increased tone significantly above 100% when nerves were functional (top panel) but reduced it slightly when nerve activity was abolished by tetrodotoxin (106 M) in the bottom panel. , p < 0.05; , p < 0.01 compared with 100% (n = 5).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Effects if ODQ (105 M) added after TEA (20 mM) in the presence of nerve function (top panel) and in the absence of nerve function (bottom panel). TEA increased tone in the presence and in the absence of nerve function, in contrast to ODQ (Fig. 1). However, the three values obtained in the presence of active nerves were not quite significantly different when tested by one-way ANOVA (p = 0.061), but the values obtained after block of nerve function were highly significantly different (p = 0.0003). ODQ after TEA had no further effect with or without nerve function. , p < 0.05; , p < 0.01 compared with controls (n = 7).

Drug Effects on Relaxation to EFS. ODQ (10⁵ M) markedly reduced relaxation to EFS (40 V/cm, 5 pps, 0.3-ms duration), converting the relatively rapid relaxation to a phasic contraction followed by a small, slow relaxation (Fig. 3, B and C) not further affected by L-NAME (Fig. 3C). The inhibition of relaxation is summarized in Table 2. Figure 4 shows that ODQ, in contrast to L-NOARG (3 × 10⁴ M), reduced but did not abolish relaxation to EFS. In contrast to ODQ and confirming previous findings (Daniel et al., 2000), 20 mM TEA had no effect to inhibit the amplitude of relaxation when present alone (Fig. 5), although it did reduce the extent of relaxation in some experimental series (Table 2). However, when TEA was present together with ODQ, relaxation to EFS was abolished (Figs. 3D and 5), as it was with L-NOARG.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Representative tracings from experiments in which effects of ODQ were examined. All traces show baseline tone before applying 2 g of tension (not shown), tone development, responses to EFS, effects after 20 min on tone and response to EFS of drugs, if any were applied, then relaxation to sodium nitroprusside (104 M) after 20 min or more, and, finally, subsequent relaxation to calcium-free solution with 0.1 mM EGTA. A, control recording. Both the tone and the relaxation response to EFS was stable over time. Note that relaxation to sodium nitroprusside relaxed nearly to baseline (passive) tension. B, effect of ODQ (105 M). In the absence of 106 M tetrodotoxin, it increased tone and changed the response to EFS from mono- or biphasic relaxation to phasic contraction followed by a smaller slower relaxation and recovery. The relaxation to sodium nitroprusside was partially inhibited compared with control. C, effect of ODQ followed by L-NAME (3 × 104 M). The effects of ODQ on tone, EFS, and sodium nitroprusside responses were not further changed after L-NAME. In this experiment, but not all, the relaxation to calcium-free EGTA solution was reduced (compare with B). D, effect of 20 mM TEA followed by ODQ. Note that TEA enhanced tone but did not inhibit relaxation to EFS. Previous studies (Daniel et al., 2000) showed that it reduced relaxation to sodium nitroprusside about 50%. Subsequent ODQ now abolished all responses to EFS and nearly abolished responses to sodium nitroprusside. Subsequent relaxation to calcium-free solution with EGTA solution was complete.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Comparison of the effects of ODQ (105 M) and L-NOARG (3 × 104 M) on the nadir of relaxation to EFS. Both raised the nadir, but L-NOARG raised it to values not different from 100% (no relaxation), whereas ODQ raised it significantly but not to 100%. , p < 0.01, compared with controls (n = 5, 4).

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Relaxation after 20 mM TEA and after subsequent ODQ (105 M). Results expressed in terms of the magnitudes of relaxation instead of the nadirs of relaxation (Table 2). After TEA, which increased tone, relaxation was increased, but following the addition of ODQ, it was abolished (not significantly different from 0). One-way ANOVA gave p < 0.0001 (n = 7).

Drug Effects on Relaxation to Sodium Nitroprusside and Calcium-Free (EGTA) Solution. L-NOARG had no effect (Fig. 6 and Table 3) on relaxation to sodium nitroprusside (3 × 10⁴ M), irrespective of the presence or absence of tetrodotoxin (not shown). ODQ or TEA each reduced relaxation to sodium nitroprusside, and together they markedly reduced it but did not abolish it (Figs. 3D and 7), unrelated to the presence or absence of tetrodotoxin (not shown). In most cases, the residual tone (assumed to be passive) after EGTA was the same irrespective of any pretreatment (Fig. 6 and Table 4). However, when tetrodotoxin was present, the combination of ODQ and TEA significantly increased it (Fig. 7).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Comparison of the effects of ODQ and L-NOARG on nadirs of relaxation to sodium nitroprusside (104 M) and to calcium-free EGTA (0.1 mM). Previous studies showed that L-NOARG has no effect on sodium nitroprusside-induced relaxation (Daniel et al., 2000). However, as shown in the top panel, ODQ increased the nadir of relaxation to sodium nitroprusside significantly, compared with L-NOARG. It did not significantly affect the nadir to removal of extracellular calcium as shown in the bottom panel. , p < 0.05 unpaired comparison (n = 6, 5).

View this table: [in this window] [in a new window]   TABLE 3 Tone nadir after sodium nitroprusside (104 M) Initial tone = 100%. Means ± S.E.M.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Nadirs of relaxation to sodium nitroprusside (top panel) or calcium-free EGTA (0.1 mM) (bottom panel) after TEA and ODQ irrespective of the presence (right histogram) or absence (left histogram) of nerve activity. Although the final tone levels achieved after sodium nitroprusside were not significantly different from 100% initial tone in the presence or absence of nerve activity, there was a small relaxation since TEA had increased tone from the initial level (Fig. 2). The bottom panel shows that relaxation to calcium-free solution was also impaired significantly by TEA with ODQ when tetrodotoxin was present. , p < 0.01 paired comparison (n = 7).

Patch-Clamp Study. ODQ (10⁵ M) had no effect on outward currents when the patch pipette had 200 nM free Ca²⁺ (Fig. 8) This is in contrast to effects of TEA (20 mM) or L-NOARG (3 × 10⁴ M), shown previously (Salapatek et al., 1998c) and confirmed here (not shown) to inhibit them by about 80% at maximum depolarization. We also reconfirmed that sodium nitroprusside (10⁴ M) restored the currents inhibited by L-NOARG (Salapatek et al., 1998c).

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Current/voltage plots of the lack of effect of 105 M ODQ on outward currents induced by depolarizing steps (20 mV, 250 ms) from 50 to + 90 mV. Either L-NOARG or TEA reduced these currents by 70 to 80% (not shown), confirming previous results (Salapatek et al., 1998c).

	Discussion

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ODQ is a widely used inhibitor of cytosolic guanylate cyclase (Ignarro et al., 1999; Chang et al., 2000; Gorodeski, 2000a,b; Janssen et al., 2000; Kwan et al., 2000; Tseng et al., 2000; Yao et al., 2000). In this study 10⁵ M ODQ increased tone and raised the nadir of NO-mediated relaxation (Jury et al., 1992) to nerve stimulation (EFS) from 38.5 to 77.5% of initial tone and changed the response qualitatively. Instead of a large fast relaxation, there was a small initial phasic contraction followed by a slow, small relaxation (Fig. 3, B and C). This relaxation was unaffected by subsequent L-NAME, suggesting that it is not NO-mediated.

After tetrodotoxin abolished responses to EFS, the tone increase to ODQ and the altered EFS response following ODQ were also eliminated, suggesting that both were nerve-mediated. Reduced responses to basal release of neural NO, acting either on interstitial cells of Cajal (ICC) or on nerve endings, likely accounts for the tone increase after ODQ (see discussion below). Previous studies (Allescher et al., 1988) showed that stimulation of intrinsic nerves of LES releases acetylcholine as well as NO. Acetylcholine was the probable mediator of contraction, observed when actions of neural NO were reduced by ODQ. It also may have contributed to the increase in tone when ODQ was administered with nerves active. Although not mediated by NO, the residual relaxation after ODQ involves opening of K⁺ channels, since it was abolished when TEA was present together with ODQ.

We did not evaluate the efficacy of ODQ to block elevations of cGMP levels, so it may have inhibited some responses nonselectively, or some tissue components may have resisted its effects. However, it selectively inhibited two NO-mediated responses (relaxation to EFS and to sodium nitroprusside). Thus, it is unlikely that the failure to inhibit NO actions when NO was derived from muscle can be explained by resistance to ODQ. It did increase spontaneous tone when nerves were functioning, but we attribute this effect to inhibition of the action of NO released from nerves under basal conditions because it disappeared after tetrodotoxin. Thus, its actions appeared efficacious against some NO-mediated events and selective in that it failed to act on other responses.

We postulate that ODQ eliminates most or all of the effects of NO released from nerves, disinhibiting (Fox-Threlkeld et al., 1999) and/or unmasking the response to the release of acetylcholine previously obscured by NO actions. We speculate that nerves release two inhibitory mediators, one NO and the other released in response to NO release. Dependence on NO release would explain why L-NAME alone usually completely inhibits relaxation to low-frequency (1-5 pulses per second) EFS (Jury et al., 1992). According to this model, neural NO acts on cGC to increase cGMP levels, but the other mediator acts to open K⁺ channels. The combination of ODQ and TEA would, on either model, inhibit both the NO/cGC and the K⁺ channel-mediated components of the response.

NO released from nerves may act on cGC in muscle or on the ICC. ICC are present in canine LES, intercalated between nerve endings and muscle to which they are connected by gap junctions (Allescher et al., 1988). Similar structural relations are found in other species and parts of the gastrointestinal tract (e.g., Daniel and Posey-Daniel, 1984; Ward et al., 1998; reviewed in Sanders et al., 1999). Recent studies in mutant W/W^V mice with apparently normal nitric-oxide synthase innervation of the LES, but lacking intramuscular ICC (Ward et al., 1998), found that nerve stimulation did not induce the usual relaxation and hyperpolarization from NO release and that NO donors also became less effective to induce relaxation and hyperpolarization in the absence of ICC. This dependence of NO-mediated neural effects on ICC was also found in the pylorus and gastric fundus (Burns et al., 1996; Ward et al., 1998). Thus, NO released from nerves may activate cGC in ICC rather than in smooth muscle. The NO-mediated activation of cGC may produce hyperpolarization in ICC cells, which is transmitted to LES muscle by gap junctions or by other means. Studies of responses of intramuscular ICC to NO are lacking because of the technical difficulty of isolating them. However, inhibitors of gap-junction conductance did not interfere with nerve-mediated relaxation of the LES (Daniel et al., 2001b). LES gap junctions had connexins 43 and 40 colocalized in them (Wang and Daniel, 2001), and the conductance properties of such junctions are unknown.

Ionic mechanisms involved in neural NO effects are incompletely understood. Previous studies showed that NO-mediated hyperpolarization accompanied relaxation of muscle and appeared to be associated with current flow through K⁺ channels (Christinck et al., 1991; Jury et al., 1992; Cayabyab and Daniel, 1995). Although 20 mM TEA alone had no inhibitory effect on amplitudes of nerve-mediated relaxation, it reduced outward currents ~80% in LES cells (Salapatek et al., 1998c). Whether LES relaxation after TEA was still associated with hyperpolarization has not been determined. The sites of K⁺ channels, ICC or muscle, affected by NO from nerves or the secondary inhibitory mediator are unclear. Clearly, the mechanisms by which LES relaxation and hyperpolarization are induced after activation of intrinsic nerves are complex and need further study.

Previous studies showed that activation of BK_Ca channels driven by release of NO from a membrane-bound nitric-oxide synthase accounted for large outward currents, cellular hyperpolarization, and reduced tone in canine LES (Salapatek et al., 1998b,c). These currents were inhibited ~80% by L-NOARG, iberiotoxin, or TEA. They were restored by sodium nitroprusside after L-NOARG but not after the K⁺ channel blockers. The nitric-oxide synthase activity depended upon Ca²⁺ entering through L-type calcium channels, closely associated with the nitric-oxide synthase in caveolae (Daniel et al., 2001a). The tone modulation from nitric-oxide synthase activity in LES strips was abolished by L-NOARG and 20 mM TEA but only partially by iberiotoxin (Daniel et al., 2000). This suggested that additional K⁺ channels besides BK_Ca were activated by NO of myogenic origin.

ODQ had no effect on tone in the presence of tetrodotoxin and no effect on outward currents. Thus, there appears to be no role for cGC in the modulation of tone and enhancement of outward currents by NO from myogenic nitric-oxide synthase. We did not test that cGC was still active in isolated LES cells. However, the actions of NO to drive outward currents do not require its activity. L-NOARG reduces the outward currents by 80% and NO donors restore them. NO in other systems also operates independently of cGC (Ahern et al., 1999; Ignarro et al., 1999; Pinilla et al., 1999; Taglialatela et al., 1999; Garry et al., 2000; Janssen et al., 2000; Liu et al., 2000; Mazzuco et al., 2000; Takeda et al., 2000; Tseng et al., 2000).

NO from the muscle membrane may act locally on nearby ion channels; examples of such local actions have been reported (Bolotina et al., 1994). The NO-dependent outward currents depend on Ca²⁺ entry through L-type calcium channels (Salapatek et al., 1998b,c). However, in opossum LES Akbarali and Goyal (1994) found that NO inhibits L-type calcium currents. Nifedipine abolishes tone in canine LES. However, inward currents opened by depolarization and carried by L-type calcium channels could not be demonstrated in LES cells from dog or opossum, even after all potassium currents were blocked (Jury and Daniel, 1996; J. Jury and E. E. Daniel, unpublished data), but they were easily demonstrated in the body of opossum esophagus. Yet, as noted above, NO-driven BK_Ca currents in LES are inhibited when L-type calcium channels are blocked. The presence of these channels in close proximity to nitric-oxide synthase in caveolae (Daniel et al., 2001a) may result in unexpected properties, including altered voltage dependence and opening properties.

Relaxation to sodium nitroprusside, a source of exogenous NO, was reduced by TEA or by ODQ. Together, they nearly eliminated it. This implies that exogenous NO acts by both activating cGC and opening K⁺ channels independently of cGC. Since ODQ had no effects on NO-driven outward currents in LES cells, TEA likely inhibited exogenous NO actions at sites on which myogenic-derived NO acts, and ODQ inhibited them on sites at which NO from nerves acts, possibly in ICC. Exogenous NO had access to both sites. In a previous study (Daniel et al., 2000) we found that TEA induced a tone increase when nerve function was abolished and occluded the tone increase to L-NOARG, consistent with an action of TEA to block effects of myogenic NO release. However, cGC was not demonstrated to be present or responsive to NO in isolated LES cells. If it is absent or nonresponsive to NO in LES cells in situ but present in ICC, this would provide an explanation for the differential effects of ODQ in vitro noted above.

In summary, these findings show that relaxation of the canine LES by neural NO is more complex than originally presumed; i.e., NO from nerves appears to activate cGC, likely in ICC, whereas NO from muscle apparently does not act through cGC. NO from muscle activates K⁺ channels, whereas NO from nerves causes relaxation, which does not require opening of TEA-sensitive potassium channels. NO added exogenously acts through both ODQ- and TEA-sensitive mechanisms.