Myogenic NOS in canine lower esophageal sphincter: enzyme activation, substrate recycling, and product actions

Anne Marie F. Salapatek, Yu-Fang Wang, Yu-Kang Mao, Masataka Mori, and Edwin E. Daniel

Playfair Neuroscience Unit, Toronto Hospital (Western Division), Toronto M5T 2S8; Department of Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; and Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 862, Japan

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Depolarization elicited outward K+ currents from canine lower esophageal sphincter (LES) muscle cells, primarily through iberiotoxin (IbTX)- and tetraethylammonium-sensitive Ca2+-dependent K+ channels. Current magnitudes varied with pipette Ca2+ concentration (EC50 = 108.5 nM). NG-nitro-L-arginine (L-NNA, 10-4 M), IbTX (10-8 M), or buffering intracellular Ca2+ to 8 nM decreased outward currents >80%. Sodium nitroprusside (NaNP, 10-4 M) restored L-NNA-inhibited or low intracellular Ca2+ concentration (not IbTX)-inhibited currents. L-NNA or IbTX application depolarized LES cells from -43 to -35 mV. NaNP restored the membrane potential to -46 mV after L-NNA but not after IbTX application. Nifedipine (30 µM) reduced outward currents and abolished or reduced L-NNA or NaNP effects, respectively. Immunocytochemistry revealed the presence of both argininosuccinate synthetase and argininosuccinate lyase in LES muscle cells. L-Citrulline, like L-arginine, reversed L-NNA inhibition of outward currents; only L-arginine reversed inhibition of outward currents by an antibody to argininosuccinate synthetase. Therefore, endogenous nitric oxide production, activated by Ca2+ entrance involving L-type Ca2+ channels, may continuously enhance outward currents to modulate LES muscle cell membrane potential and excitability.

nitric oxide synthase; calcium-dependent potassium channel; L-citrulline recycling; lower esophageal sphincter tone; sarcoplasmic reticulum; smooth muscle

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

WE HAVE RECENTLY demonstrated that a constitutive, Ca2+/calmodulin-dependent and membrane-bound nitric oxide synthase (cNOS) is present in membranes of smooth muscle cells of the canine lower esophageal sphincter (LES) (41, 43), and we obtained preliminary evidence for a myogenic NOS in other sphincters along the gastrointestinal tract (unpublished observations). We found morphological/histochemical and functional/biochemical evidences for a myogenic NOS, dependent on Ca2+ entry for activity and acting to open K+ channels to modulate contraction (41, 43). However, the LES NOS has structural differences from other cNOS isoforms, since some antibodies raised to neural, endothelial, and inducible forms of cNOS did not immunostain LES NOS (Ref. 43 and unpublished observations), similar to findings for rabbit gastric muscle NOS (33, but see Ref. 47). In this study, evidence derived from single LES smooth muscle cells demonstrates both existence and function of cNOS and the regeneration of the NOS substrate L-arginine (L-Arg). Furthermore, the mechanism of activation of myogenic NOS and the mode of action of its product, nitric oxide (NO), are explored in isolated smooth muscle cells from this gastrointestinal region.

Much work on LES musculature has demonstrated that the LES in many species has the ability to tonically contract or display tone (summarized in Ref. 11). LES tone is dependent on extracellular Ca2+, since tone can be reduced or abolished with plasma membrane Ca2+ channel blockade by dihydropyridines or removal of external Ca2+ (1, 5, 14, 39-41). We hypothesize that high intracellular Ca2+ maintained by continual Ca2+ influx through L-type Ca2+ channels continuously activates canine LES cNOS, resulting in an ongoing release of NO that restricts contraction. Salapatek et al. (43) showed that blockade of LES NOS resulted in persistent contraction of muscle strips as predicted by this hypothesis. Murthy et al. (32, 33) demonstrated in rabbit gastric smooth muscle cells that vasoactive-intestinal polypeptide acted via membrane receptors to activate a G protein, causing Ca2+ influx and myogenic NOS activation. Increasing intracellular Ca2+ levels by other means such as KCl, cholecystokinin octapeptide, or acetylcholine perfusion, in the presence of a protein kinase inhibitor, stimulated L-[3H]citrulline production, indicative of NOS activation (33). In LES cells, myogenic NOS may be continuously activated by high intracellular Ca2+ concentrations ([Ca2+]i) maintained by ongoing Ca2+ influx and/or release of Ca2+ from intracellular storage sites such as the sarcoplasmic reticulum (SR) and may not require a receptor-mediated event (38, 39).

Ongoing LES NOS activity would result in continual release of its product, NO. The mechanism of action of this endogenous NO or applied NO has not been established. Many studies suggest that NO hyperpolarizes smooth muscle by opening K+ channels [Refs. 9, 10, 23, 26, 27, and 33 and reviewed by Sanders and Ward (44)]. Activation of K+ channels or currents has been demonstrated in feline and opossum esophageal muscle (9, 10, 13, 22, 23, 31). A number of studies conducted on a variety of smooth muscles (4, 6, 7, 27) including recent studies on esophageal smooth muscle (23, 31) suggest that NO activates Ca2+-dependent K+ channels (KCa) as well as other K+ channels (43). Previous studies showed that NO plays an important role in LES functioning. For example, inhibition of neural NOS (nNOS) abolished the inhibitory junction potential (IJP) and the relaxation of LES to electric field stimulation (EFS) in many species (9, 10, 12, 13, 22, 35, 46, 48). Also, application of exogenous NO or NO donors mimics EFS effects by relaxing and hyperpolarizing the smooth muscle of the LES (9, 13, 22, 23, 36).

If canine LES has a continually activated cNOS, the enzyme must have a continuous supply of substrate. L-Arg could be continuously supplied to LES cells in three ways. 1) Extensive L-Arg stores exist in LES smooth muscle cells or another cell type that supplies LES cells by transport using a basic amino acid transporter, as can occur in neurons (50). However, isolated LES smooth muscle strips or cells demonstrate ongoing NOS activity for hours (43), despite the fact that no L-Arg is added to the extracellular solution in our preparations. 2) L-Arg is supplied by de novo synthesis in LES muscle cells. However, no evidence of this capability exists (29). 3) L-Arg is being regenerated from L-Cit, a NOS reaction product, within the LES smooth muscle cell. Two urea enzymes, argininosuccinate lyase (AL) and argininosuccinate synthetase (AS), can perform this conversion [reviewed by Morris (29)]. These enzymes have been demonstrated in liver and kidney tissues (29) as well as in vascular smooth muscle (19) and canine enteric neurons (45). The result of the combined actions of AS and AL enzyme activity within LES smooth muscle cells would be the regeneration of the NOS substrate, L-Arg, from L-Cit; the latter is the other NOS product in addition to NO. The AS enzyme catalyzes the conversion of citrulline with aspartate to argininosuccinate (19). This conversion step is considered to be rate limiting (28).

The objectives of this study were fourfold: 1) to characterize the ionic currents from isolated LES smooth muscle cells using whole cell patch-clamp recording, 2) to test for the functional presence of NOS activity by inhibiting it and to define the mechanism of NOS activation and its consequences, 3) to test the mode of action of exogenous (applied) or endogenous NO, and 4) to investigate whether L-Arg resynthesis from L-Cit occurs to allow continuous NO production. Preliminary reports of this work have been presented (42, 43).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Patch-Clamp Studies

Cell isolation. Fasted mongrel dogs of either sex were euthanized with an overdose of pentobarbital sodium (100 mg/kg), following a protocol approved by the McMaster University Animal Care Committee and following the guidelines of the Canadian Council on Animal Care. The gastroesophageal region was excised and stored in cold Krebs-Ringer solution equilibrated with 5% CO2 and 95% O2 and having the following composition (in mM): 115.0 NaCl, 4.6 KCl, 1.2 NaH2PO4, 1.2 MgSO4, 22.0 NaHCO3, 2.5 CaCl2, and 11.0 glucose. The gastroesophageal junction was opened on the gastric greater curvature side, and the mucosae were removed by blunt dissection. The muscular equivalent of the LES was revealed as a thickened ring of muscle composed of clasp fibers with oblique gastric sling fibers on either side (15). LES muscle was consistently cut from the clasp region of the LES because functional differences have been demonstrated among the different muscle bundles of the LES in a number of species including humans (36), and circular muscle strips from the clasp fibers region were dissected out (1, 15). Smooth muscle strips were cut into squares of ~2 mm3. Smooth muscle squares were incubated in a dissociation solution containing (in mM) 125.0 NaCl, 10.0 glucose, 5.0 KCl, 1.0 CaCl2, 1.0 MgCl2, 10.0 HEPES, and 2.5 EDTA salt (pH 7.2) to which was added one of three collagenase blends (F, H, or L; 130 mg/ml), papain (130 mg/ml), (-)-1,4-dithio-L-threitol (15.4 mg/ml), and BSA (100 mg/ml) and then incubated at 37°C for 1 h [a modification of the isolation method used by Janssen and Sims (20)]. Tissues were washed with enzyme-free dissociation solution and then mechanically agitated with siliconized Pasteur pipettes to disperse tissue and isolate single smooth muscle cells. All cells used in this study were patch clamped at room temperature (22-24°C) within 8 h of isolation.

Recording techniques. Isolated LES smooth muscle cells were allowed to settle and adhere for 30 min to the bottom of a recording chamber that was mounted on an inverted microscope. The cells were then washed by perfusion with Ca2+-containing external solution (in mM: 140.0 NaCl, 4.5 KCl, 2.5 CaCl2, 1.0 MgCl2, 10.0 HEPES, and 5.5 glucose, pH adjusted to 7.35 with NaOH). Patch electrodes were made with borosilicate glass capillary tubes using a Flaming Brown micropipette puller (Sutter Instruments). Pipettes were polished using a microforge (Narishige MF-83). In all experiments, unless otherwise stated, pipettes were filled with a standard high K+-Ca2+ solution (pipette Ca2+ concentration = 1,000 nM) containing (in mM) 140.0 KCl, 0.5 CaCl2, 1.0 MgCl2, 10.0 HEPES, 4.0 Na2ATP, and 0.3 EGTA, pH adjusted to 7.2 with KOH. At times, the Ca2+ content of the standard pipette solution was modified by altering the CaCl2 and EGTA content (pipette KCl concentration was then altered accordingly to maintain osmolarity) to achieve 8, 50, 200, and 8,000 nM Ca2+ in the pipette solution. Solution Ca2+ concentrations were calculated using MaxChelator software (version 6.72) (3). To block all K+ flux, KCl was substituted with 122 mM CsCl and 22 mM tetraethylammonium (TEA) in the standard pipette solution. Pipette tips were fire polished to resistances of 3-6 MOmega . The current flow between the pipette and the bath solution was compensated to achieve a zero baseline before seal formation. Standard tight-seal recording techniques for whole cell recording were employed (18). Briefly, after gigaseal formation, access to the interior to the cell was obtained by further suction and whole cell recordings were made.

Membrane currents were obtained in response to depolarizations from a -50 mV holding potential. Currents were measured with an Axopatch-1C patch-clamp amplifier (Axon Instruments), filtered with a Bessel filter (-3 dB at 1 kHz), and recorded online by a computer (IBM AT) using pCLAMP software (Axon Instruments, version 5.5). No capacitance compensation or leak subtraction was performed; however, whole cell capacitance and access resistance were routinely recorded. Currents were elicited by one of three voltage protocols, two of which involved incremental 20-mV depolarizations that were held for 200 ms from either -100 to +80 mV or -50 to +70 mV. For these two protocols, current-voltage curves were constructed for steady-state currents (at the end of each current trace) at each voltage step. The third voltage protocol, a ramp protocol, was also performed from a holding potential of -50 mV, and then the voltage was continuously increased from -60 to +80 mV over 1 s. The peak outward current was then measured and compared after each pharmacological manipulation. LES smooth muscle cells could be patch clamped for up to 2 h in normal external solution with no significant change in cell currents or membrane potential (data not shown). In traces displayed in Figs. 1-3, 5, and 8, the currents were derived from single illustrative traces unless otherwise stated.

Immunohistochemistry

Tissue preparation. Three dogs were euthanized, and strips of LES were prepared as described above. LES strips were then fixed overnight in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C. The fixed tissues were then washed with 0.1 M phosphate buffer. Tissues to be used for cryostat sectioning were cut into small pieces and then stored in 30% sucrose in 0.1 M phosphate buffer for cryoprotection at 4°C for 24 h; they were then sectioned at 15 µm thickness in a cryostat (Leitz 1720 digital). The cells or sections were collected on glass slides coated with gelatin.

Single immunohistochemical staining. Whole mount preparations and cryostat sections were incubated overnight at 4°C in rabbit anti-sera raised against AS or AL enzymes and purified from rat AS and AL enzyme or in nonimmune rabbit serum. Sera were applied at a dilution of 1:500 for AS antiserum and 1:100 for AL antiserum. Yu et al. (51) have described the details on antibody preparation. The antisera were washed from the sections and preparations with phosphate buffer. Both antibodies were visualized with FITC-labeled goat anti-rabbit IgG (Jackson ImmunoResearch) diluted at 1:50. After they were washed with PBS, the sections and preparations were then mounted in 80% glycerol in PBS (pH 10) and viewed on a Leitz microscope equipped with fluorescence epiluminator and N2 filter. Kodak T-MAX 400 film was used for black-and-white photography. As a control for nonspecific staining, the primary antibody was omitted, resulting in lack of staining. Nonimmune rabbit serum used as primary antibody did not lead to staining.

Double immunohistochemical staining. To examine whether any of the neurons showing immunoreactivity for AS also contained nNOS, a double immunohistochemical staining procedure was performed. The tissues were incubated overnight at 4°C in complex antiserum solution of rabbit AS and guinea pig nNOS. After excess antibody was washed out with PBS, tissues were incubated in goat anti-rabbit FITC IgG (1:50) plus rhodamine Lissamine rhodamine sulfonyl chloride (LRSC)-labeled donkey anti-guinea pig IgG (Jackson ImmunoResearch) (1:50) for 60-120 min at room temperature and stored overnight at 4°C. After washing with 0.1 M PBS, the tissues were mounted in 80% glycerol in PBS. Sites of immunoreactivity of both AS and nNOS were visualized with a microscope equipped with an I2 filter for rhodamine LRSC and an N2 filter for FITC. Black-and-white photography was performed as described above.

Immunohistochemical staining of isolated LES smooth muscle cells. LES smooth muscle cells were isolated as described above and fixed with 4% paraformaldehyde in 0.1 M PBS for 20 min at room temperature. After cells were washed in PBS by centrifugation at 3,000 rpm (3 × 15 min), cells were incubated in AS or AL antibodies overnight at 4°C and then incubated in FITC-labeled goat anti-rabbit IgG secondary (1:50). This protocol always results in some cells being completely isolated and some cells remaining in clumps after centrifugation. After the immunoreaction was stopped by washing in PBS with centrifugation (3 × 15 min), individual cells and clumps of cells were mounted in a mounting medium (as described above) on a glass slide and viewed under the fluorescent microscope.

Data Analysis

Data are expressed as arithmetic means ± SE. The letter n indicates the number of experimental animals. Multiple observations for the same manipulation (pharmacological and/or electrophysiological) made on cells from one animal were averaged. The data sets involving the effects of channel blockers, NOS blockade, and NO liberators on the peak steady-state current or at peak voltage applied were analyzed by one-way ANOVA. The statistical significance of differences in the means was determined by Tukey-Kramer multiple comparisons test in which P values <0.05 were considered significant. EC50 values were obtained using Fig.P software, version 6.0 (Durham, NC) using a four-parameter-fit model.

Solutions and Drugs

Test solutions were applied by perfusion (2-3 ml/min) or added to the bath to get the final concentration as indicated. Drug concentrations applied were chosen on the basis of other studies done in our laboratory that showed them to be maximally effective. Stock solutions of NG-nitro-L-arginine (L-NNA) and cystamine were made up in 0.1 N HCl and stored at -22°C initially and diluted further (100- to 1,000-fold) with extracellular solution. Iberiotoxin (IbTX), a generous gift from Dr. I. Rodger (Merck Frosst Canada), was dissolved in double-distilled H2O and stored at -22°C, with each aliquot being defrosted once and used over a 6-h study period. The antibody for the AS enzyme was applied in the standard pipette solution (described above) at a dilution of 1:500. All drugs were added to the chamber in microliter volumes, and routine controls with the vehicles used for dissolving each reagent were done to exclude nonspecific effects of the diluent. All chemicals, excluding those noted exceptions, were purchased from Sigma Chemical (St. Louis, MO).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Two hundred eleven smooth muscle cells were patch clamped in this study. Cell length ranged from 100 to 300 µm. These cells contracted reversibly to 60 mM KCl or 10-5 M carbachol, returning to their previous elongated shape when these agents were removed. Only cells with an access resistance of <25 MOmega were studied. In these experiments, access resistance ranged from 8 to 21 MOmega , and the mean whole cell capacitance measured was 58.2 ± 4.5 pF. When cells were studied with a standard high K+-Ca2+ pipette solution containing 1,000 nM Ca2+, under current-clamp conditions, the mean resting membrane potential recorded was -43 ± 2 mV (n = 6), similar to membrane potentials recorded from canine or opossum LES tissue with microelectrodes (11, 22).

Whole Cell Current Characterization

When a standard high K+-Ca2+ pipette solution was applied to freshly isolated LES smooth muscle cells and a voltage protocol was applied in which cells were held at -50 mV and depolarized by 20-mV steps from -100 to +80 mV and held for 200 ms, large, noisy, outward currents were elicited (see Fig. 1). When some cells were studied at a holding potential of -90 mV to determine if there were currents present that were inactivated at a holding potential of -50 mV, no additional currents, current magnitude differences, or kinetic changes in current traces were observed.


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Fig. 1.   Standard voltage-evoked outward currents. Inset: typical voltage-clamp recording of whole cell currents recorded from lower esophageal sphincter (LES) smooth muscle cells with a standard high K+-Ca2+ pipette solution. Cells were held at -50 mV and stepped from -100 to +80 mV in 20-mV increments and held for 200 ms (see voltage protocol pictured at top). Bottom: current-voltage curve constructed for the mean steady-state currents recorded at each voltage shows that the peak outward current recorded during the highest voltage step (+80 mV) was 3,667.9 ± 48.9 pA (n = 6).

When cells were studied under the same voltage-clamp conditions with a pipette solution in which KCl was substituted with CsCl (122 mM) and TEA (a nonspecific K+ channel blocker; 22 mM), outward currents were significantly smaller than outward currents recorded with a standard high K+-Ca2+ pipette solution at higher voltage steps as shown in Fig. 2, A-C. This result implied that outward currents were primarily carried by K+. To test this hypothesis further, the outside K+ concentration and K+ concentration inside the cell were equalized. In tail current recordings, there was a significant shift in the reversal potential (Erev) to the right (more positive): Erev = 0 ± 2 mV, n = 4, from an Erev with 4.5 mM KCl in the external solution of -82 ± 3 mV, which is close to the calculated K+ equilibrium potential (-86 mV) for these experimental conditions. These results confirmed the dependence of outward currents on K+ flow out of the cell.


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Fig. 2.   Characterization of outward currents. Typical voltage-clamp responses when cells were held at -50 mV and stepped from -50 to +70 mV in 20-mV increments and held for 200 ms (see voltage protocol pictured at top, middle) are shown. A: typical control voltage-clamp recording of LES whole cell currents using a standard pipette solution. B: typical whole cell currents recorded from another LES cell using a pipette solution in which KCl has been substituted by CsCl (122 mM) and tetraethylammonium (TEA; a nonspecific K+ channel blocker, 22 mM). Peak steady-state currents were greatly reduced. In separate cells, when a pipette solution low in Ca2+ was applied (8 nM, 11 mM EGTA), outward currents were also reduced (not shown; summarized in C). C: current-voltage curves obtained with steady-state currents measured at each voltage for each pipette solution applied (as indicated beside each curve; SE bars are shown). A pipette solution low in Ca2+ (8 nM, 11 mM EGTA; indicated by ×) significantly reduced outward currents over control levels (black-triangle) at the +50-mV step (389.1 ± 8.5 vs. 2,569.9 ± 9.7 pA, P < 0.001) and +70-mV step (491.7 ± 8.2 vs. 3,267.9 ± 8.9 pA, P < 0.001; n = 4). A pipette solution in which KCl was substituted with CsCl and TEA (square ) reduced outward currents over control levels and further than the application of a low-Ca2+ pipette, with the mean peak outward current recorded = 92.6 ± 12.1 pA at +50 mV (P < 0.001) and 123.7 ± 11.1 pA at +70 mV (P < 0.001, n = 4). D: average current-voltage curves obtained with steady-state currents at each voltage after the sequential addition of K+ channel blockers (indicated beside each curve) (n = 4; SE bars are shown). Decrease in peak outward current was not significantly reduced with apamin (black-triangle); however, currents were significantly reduced over control (square ) levels after TEA addition (×), that is, 384.6 ± 8.5 pA vs. 2,569.9 ± 9.7 pA at +50 mV and 556.8 ± 4.5 pA vs. 3,267.9 ± 8.9 pA at +70 mV (P < 0.001, n = 4).

As shown in Fig. 2C, Ca2+ chelation and reduction of intracellular Ca2+ by application of a low-Ca2+ pipette solution ([Ca2+]pipette = 8 nM, [EGTA]pipette = 11 mM) significantly reduced outward currents over control levels, suggesting that the K+ channels that opened with voltage were also Ca2+ dependent (KCa channels). For this reason, a variety of K Ca channel blockers were applied. As shown in Fig. 2D, apamin, a specific small-conductance KCa channel blocker (10-6 M) reduced outward currents insignificantly; therefore, this subset of KCa channels contributed little to the outward currents observed. However, TEA (5 mM), a nonspecific large-conductance KCa channel blocker at this concentration (2), sequentially added to the bath greatly reduced outward currents (see Fig. 2D), suggesting the involvement of large-conductance KCa channels in the outward current. As summarized in Table 1, the specific large-conductance KCa channel blocker, IbTX (10-8 M), significantly reduced outward currents recorded with a standard pipette solution, confirming that the majority of K+ outward current was conducted through voltage-sensitive, large-conductance KCa channels. In the current-clamp mode, IbTX at 10-8 M depolarized cells from -45 ± 3 mV to -38 ± 4 mV (n = 4, P < 0.05). These data suggested that IbTX-sensitive channels were contributing outward current to maintain the resting potential in these cells.

                              
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Table 1.   Effect of various pharmacological interventions on whole cell outward currents elicited by depolarization and recorded from isolated LES smooth muscle cells

Endogenous NOS Inhibition

When the NOS inhibitor L-NNA (10-4 M) was applied, the outward current recorded with standard pipette solution was significantly reduced, as demonstrated in Fig. 3A and summarized in Table 1. This finding suggested that there was ongoing myogenic NOS activity, resulting in endogenous NO release. L-NNA caused cell depolarization in all cells studied under current clamp from -43 ± 2 mV to -35 ± 1 mV (n = 6, P < 0.05). This L-NNA-induced depolarization implied that NOS activity and the resulting NO release opened K+ channels, causing LES cell membrane hyperpolarization. Conversely, the blockade of NOS and endogenous NO liberation by applied L-NNA resulted in K+ channel closure and the observed cell membrane depolarization. Single cells, when observed, always contracted after L-NNA addition, suggesting that the resulting depolarization opened voltage-sensitive Ca2+ channels that caused contraction.


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Fig. 3.   Effect and specificity of nitric oxide synthase (NOS) inhibitor actions on outward currents. Current traces recorded from 2 separate cells (A and B) using a ramp voltage protocol (pictured at top) are shown. Average data are summarized in Table 1. A: typical current trace before and after addition of the specific NOS inhibitor L-NNA (10-4 M). L-NNA significantly reduced the outward current. B: effect on outward currents of the sequential application of the NOS substrate L-arginine (L-Arg, 10-3 M) and L-NNA. There was no significant change in outward currents with the sequential application of L-NNA after L-Arg pretreatment.

The specificity of L-NNA for NOS was confirmed by the ability of L-Arg (10-3 M), a NOS substrate, to prevent L-NNA action at a higher concentration (see Fig. 3B and Table 1). L-Arg application alone apparently did not result in further NO production, since outward currents were not increased.

Mechanism of Muscular NOS Activation and Action of NO, Endogenous or Applied

Figure 4 shows the relationship between [Ca2+]pipette and the mean peak outward current magnitude. When higher [Ca2+]pipette amounts (200, 1,000, or 8,000 nM) were used, significantly higher KCa channel currents were found than with 50 and 8 nM Ca2+ in the pipette. The calculated EC50 for this relationship was 108.5 ± 1.1 nM. As shown in Fig. 5A, application of the NO liberator sodium nitroprusside (NaNP; 10-4 M) had no significant effect on outward currents when [Ca2+]pipette was high (>= 200 nM Ca2+), but currents were increased back to levels similar to those recorded with the standard high-Ca2+ pipette when a pipette with 8 nM Ca2+ was used (Fig. 5B). When the pipette contained 200 nM or more Ca2+, like L-Arg, NaNP slightly reduced outward currents (though not significantly), possibly through a negative feedback mechanism. Thus NOS appeared to be maximally activated by high [Ca2+]i, which resulted in maximal NO liberation and maximal KCa channel activation so that neither applied NO (liberated by applied NaNP) nor L-Arg could result in additional KCa channel recruitment. As summarized in Table 1, when IbTX (10-8 M) was applied to the extracellular medium, outward currents were reduced to a similar extent as by L-NNA addition. Subsequent addition of L-NNA did not significantly affect outward currents, suggesting that the myogenic source of NO acts to open KCa channels. Also, IbTX (10-8 M) blocked 75.1 ± 0.2% of the outward current restored by NaNP after L-NNA addition, confirming that NO by either the endogenous, myogenic source or the applied, NaNP-NO-liberated source acted in large part to activate KCa channels (see Table 1).


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Fig. 4.   Effect of pipette Ca2+ content on outward currents. Mean peak outward current recorded using the ramp protocol from separate cells using pipette solutions with varied Ca2+ concentrations (8, 50, 200, 1,000, and 8,000 nM) is shown; n values are 5, 5, 5, 10, and 14, respectively. Pipette Ca2+ concentrations <50 nM significantly reduced the peak outward current compared with pipette solutions containing 200 nM Ca2+ or more. Calculated EC50 for this response was 108.5 ± 1.1 nM (r = 0.999612).


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Fig. 5.   Effect of nitric oxide (NO) liberator on outward currents. Current traces recorded from 2 different cells (A and B) using the ramp protocol (pictured at top) are shown. Averaged data are summarized in Table 1. A: sodium nitroprusside (NaNP; 10-4 M), an NO liberator, had no significant effect on peak outward currents when a standard pipette solution (high K+-Ca2+) was applied. B: when a low-Ca2+ pipette solution (8 nM) was applied, peak outward currents were significantly reduced (summarized in Fig. 4). When NaNP was subsequently added, peak outward currents were significantly increased over control currents (recorded previously). Increased peak outward currents recorded with a low-Ca2+ pipette solution plus NaNP added to the bath were not significantly different from similar currents recorded with a normal high K+-Ca2+ pipette solution.

When endogenous NOS activity was blocked with L-NNA (10-4 M), NaNP (10-4 M) significantly increased the recorded outward currents when either the standard pipette solution or the pipette Ca2+ was 8 nM (see Table 1). Thus, when muscular NOS was not maximally activated, application of exogenous NO increased outward currents. However, after L-NNA, IbTX reversed NaNP restoration of outward currents. The addition of a combination of L-NNA and NaNP repolarized cells back to their resting potentials (-46 ± 2 mV; n = 6, P < 0.01). As stated above, IbTX alone depolarized LES cells and subsequent addition of L-NNA or NaNP had no further effect.

When LES cells were pretreated with nifedipine (3 × 10-7 M) for >45 min and studied with a standard pipette solution, outward currents were significantly reduced compared with controls (summarized in Table 1). Addition of L-NNA after nifedipine pretreatment did not result in any significant changes in outward currents. Thus Ca2+ entry through L-type Ca2+ channels is required for maintenance of [Ca2+]i levels sufficient for NOS activation, NO release, and subsequent KCa channel activation despite high levels of intracellular Ca2+. Furthermore, NaNP (10-4 M) added after nifedipine pretreatment (3 × 10-5 M) clearly increased outward currents but not to control levels with a standard pipette solution (56.2 ± 18.5% of control levels, Table 1). Apparently, Ca2+ entry through L-type Ca2+ channels contributed to the activation of KCa channels by NO even when there were high levels of [Ca2+]i in the general cytoplasm.

When the nonspecific guanylate cyclase inhibitor cystamine (10-3 M) was added, outward currents decreased significantly (57.7 ± 2.9%), although not to the extent that L-NNA reduced outward currents (see Table 1). This finding suggested that endogenous NO acts in part via a soluble guanylate cyclase, exerting effects on KCa channels via agents such as cGMP or cGMP-dependent protein kinase (PKG).

L-Arg Regeneration from L-Cit

Immunocytochemistry for AS and AL. To determine if canine LES cells were capable of regenerating L-Arg from L-Cit to sustain a continuous NOS activity, the presence of the necessary enzymes was first determined. When nonimmune serum from rabbits was used (Fig. 6A) or the primary antibody for AL (not shown) or AS was omitted (Fig. 7A), there was negligible nonspecific staining. However, immunoreactivity for both AS and AL enzymes occurred not only in the enteric neurons and vascular endothelia (not shown) but also in smooth muscle bundles (Figs. 6, C and D, and 7B) and cells freshly isolated from the clasp region of the LES (see Figs. 6B and 7A). Immunoreactivity for AS and AL enzymes occurred in nerve fibers as well as neurons (see Figs. 6, C and D, and 7B).


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Fig. 6.   Immunoreactivity for argininosuccinate synthetase (AS) and neuronal NOS (nNOS) enzymes. A: control cryostat section showing absence of staining when primary antibody was omitted, i.e., no exposure to serum with antibodies to AS or AL. Bar = 25 µm. B: AS enzyme immunoreactivity was present in isolated canine LES smooth muscle cells. Bar = 25 µm. Note particulate distribution of AS immunoreactivity concentrated in the periphery of cells. C and D: AS enzyme immunoreactivity was also present in ganglia of the myenteric plexus and intramuscular nerve bundles as well as canine LES smooth muscle bundles (cryostat sections). Bar (C) = 25 µm. Asterisks show AS immunoreactivity in muscle. Arrows indicate immunoreactive nerve processes. Bar (D) = 12.5 µm. E: arrows show AS immunoreactivity in some nerve cells of the myenteric plexus (Myp) in between the longitudinal muscle (LM) and the circular muscle layer (Sph), which stain much less intensely than nerves and appear unstained in this micrograph. Bar = 50 µm. F: arrows show that the same cells stained in E are also immunoreactive for nNOS. Bar = 50 µm. For A-F, n = 3.


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Fig. 7.   Immunoreactivity for argininosuccinate lyase (AL) enzyme: AL immunoreactivity of the canine LES. A: isolated cells stained with serum containing antibody to AL. Bar = 25 µm. a: Single isolated muscle cell at higher power to illustrate the particulate staining located primarily in the periphery of the cell. Bar = 12.5 µm. B: arrows indicate immunoreactive nerve bundles in LES muscle; asterisks show AL immunoreactivity in a cryostat section through muscle bundles of the LES. C: control cryostat section stained with nonimmune rabbit serum during overnight incubation. No significant nonspecific staining was observed. Bars (B and C) = 25 µm. For A-C, n = 3.

The staining for AS in LES muscle cells was particulate (see Fig. 6B) as was that for AL (Fig. 7A), similar to the staining seen previously with NADPH-diaphorase techniques in these cells (43). The time course and depth of staining in muscle cells were comparable to those of neurons and endothelia. Therefore, on the basis of morphology, LES smooth muscle cells have NOS activity (41, 43) and the enzymes, AS and AL, required for L-Arg regeneration. As shown in Fig. 6, E and F, nNOS and AS enzyme immunoreactivities were also colocalized in LES enteric neurons.

Functional evidence for L-Arg regeneration. To provide functional evidence that L-Arg regeneration was occurring and required for ongoing NO production by myogenic NOS in LES cells, we tested the abilities of L-Arg or L-Cit to reverse blockade of NO synthesis by L-NNA, resulting in inhibition of K+ outward currents, before and after inhibition of AS. Because AS enzyme activity is rate limiting for recycling of L-Cit to L-Arg, our expectation was that both L-Arg and L-Cit would restore L-NNA-reduced outward currents when AS was functioning but that only L-Arg would be effective when AS was inhibited by an antibody to AS. Figure 8A shows the typical (27) large L-NNA-sensitive outward current elicited by membrane depolarization when isolated LES smooth muscle cells were studied under whole cell voltage patch clamp. Table 2 contains a summary of these experiments.


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Fig. 8.   Effect of L-citrulline (L-Cit) or L-arginine (L-Arg) on NG-nitro-L-arginine (L-NNA) or AS antibody response. A: typical whole cell current trace obtained using a ramp protocol (pictured at top) and the sequential application of the NOS inhibitor, L-NNA (10-4 M), and L-Cit (2 mM), a NOS coproduct. See Table 2 for mean current values recorded (n = 4). B: typical, sequentially recorded whole cell current traces obtained using a ramp protocol (see top) and recorded with a pipette solution containing an AS antibody (1:500 dilution) over time (see i-iii); L-Arg (2 mM, a NOS substrate) was then subsequently added to the bath (see iv). Sequential application of L-Arg increased outward currents significantly to levels not unlike initial levels (n = 4). In separate cells, when L-Cit (2 mM) was applied after AS enzyme antibody application in the pipette solution, rather than L-Arg, outward currents continued to decline but not significantly (trace not shown, n = 3). See Table 2 for mean current values recorded under these conditions.

                              
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Table 2.   Effect of endogenous NOS manipulation on outward currents

As expected if L-Arg regeneration was occurring, L-Cit (2 mM) reversed NOS inhibition by L-NNA (Fig. 8A and summarized in Table 2), as did L-Arg. When the antibody to the AS enzyme was applied directly within the LES smooth muscle cell via the pipette solution in the whole cell patch configuration, outward currents were significantly reduced over time (Fig. 8B). As shown in Fig. 8B and summarized in Table 2, within 15 min outward currents were 39.0 ± 0.5% of the initial outward current recording [Fig. 8B, compare trace labeled AS-Ab (0 min) vs. trace labeled AS-Ab (15 min)]. Subsequent addition of NOS substrate, L-Arg (2 mM), increased outward currents to levels not different from initial levels. However, in the presence of the AS antibody, as shown numerically in Table 2, application of L-Cit (2 mM) [which gained access to the cell interior to regenerate L-Arg (demonstrated by data in Table 2)] was not able to restore NO production and its associated outward currents. When KCa channels were initially blocked with IbTX, the AS enzyme antibody or excess L-Arg had no further effect (Table 2). This result is consistent with the hypothesis that the ion channels being indirectly inactivated by the action of the AS antibody on NOS substrate levels, as well as those subsequently being activated by L-Arg addition, were indeed KCa channels, as judged by their pharmacology.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

To achieve the aims of our studies, it was first necessary to isolate viable smooth muscle cells from the canine LES and to characterize the major components of voltage-induced currents. Isolated cells had resting membrane potentials (-43 ± 2 mV) similar to those previously recorded in muscle strips with microelectrodes (22), and they were able to contract and then relax when contractile agents were applied and then washed out. Study of tail-current Erev and outward currents after ion substitution demonstrated that the major ion carrying the outward current was K+ (see text). Substitution of KCl in the pipette solution with CsCl and TEA greatly reduced outward currents, which confirmed that outward currents were primarily carried by K+. Because the resting membrane potentials of these cells were far from the K+ equilibrium potential of -86 mV, there must have been inward currents opposing K+-mediated outward currents at rest. The nature of these currents has not been explored; however, it seems likely that Cl- and Ca2+ currents contributed to inward currents at the resting potential of these cells. With the pipette Cl- concentration used in these studies, the equilibrium potential for this ion was near 0 mV. Thus any outflow of Cl- would have balanced some of the K+ outflow and contributed to a membrane potential far from -90 mV. Other studies in our laboratory showed that active tension generated by LES muscle strips was abolished by nifedipine (39, 40). Whatever the nature of inward currents, their increase in magnitude on depolarization was small relative to the increase in outward K+ currents as shown in Fig. 2 and subsequently in Figs. 3, 5, and 8.

The voltage-dependent outward currents included large-conductance KCa channel currents that were similar to the large, noisy, and sustained currents of LES cells in outward currents observed by others on esophageal body smooth muscle cells from the opossum (23, 31). Although smaller in mean current amplitude, outward currents in cells from the esophageal body also activated at potentials greater than -30 mV. Most K+ channels in LES cells were not apamin sensitive, since this blocker, specific for small-conductance KCa channels, reduced outward currents insignificantly. Application of the specific large-conductance KCa channel blocker IbTX reduced outward currents from LES cells (82.1 ± 2.7% reduction over control levels) and to levels similar to those found with the nonspecific KCa channel blocker TEA (82.9 ± 0.1% reduction over control) or when 8 nM Ca2+ was in the pipette (79.7 ± 2.6% reduction). As discussed below, it is unclear whether the use of pipettes with 8 nM Ca2+ affected outward currents by lowering Ca2+ levels near the KCa channels or by reducing cNOS activity.

Substitution experiments with CsCl and TEA reduced outward currents markedly (96.2 ± 0.1% reduction compared with control values with a standard pipette solution) and more than did IbTX. Therefore, other K+ currents were also likely present and may have contributed to NO-induced effects. Carl (8) has shown that outwardly rectifying currents exist in circular muscle of canine colon, and Koh et al. (26) showed that NO activated K+ currents in these cells in addition to those through KCa channels. Tissue strips with NO release from nerves blocked contracted tonically when NOS was inhibited by L-NNA, presumably because endogenous NO production modulated tone through effects of outward currents. IbTX also caused contraction, but subsequent L-NNA caused additional contraction (Ref. 43 and A. M. F. Salapatek, A. Lam, and E. E. Daniel, unpublished observations). In that study, a combination of nonspecific K+ channel blockers, TEA and 4-aminopyridine (4-AP), expected to block all K+ channels except possibly inward rectifiers (17, 25, 37), did cause a contraction that precluded any further contraction to L-NNA. Presumably, they blocked all channels, which contributed to the modulating effects of endogenous myogenic NO on contractile function.

Our patch-clamp studies may have missed K+ channels that rapidly inactivated during our ramp protocol, unlike KCa channels (26, 27). Moreover, we may also have missed temperature-dependent K+ channels, since the experiments with tissue strips were conducted at 37°C, whereas those with single cells were conducted at room temperature. Additional experiments on strips have shown that canine LES tissues studied at 23°C develop tone, relax to EFS, and, after nerve blockade, undergo marked further persistent increases in tone to L-NNA and TEA application (E. E. Daniel, A. Lam, and A. M. F. Salapatek, unpublished observations). Although other non-Ca2+-dependent K+ channels may have contributed an insignificant component of the outward currents recorded in this study but functioned to hyperpolarize LES cells in the muscle bath, there was no fundamental difference in function of myogenic cNOS at 23 and 37°C; endogenous myogenic NO was produced to activate TEA-sensitive channels and modulate tone.

No transient, 4-AP-sensitive K+ currents were observed even when cells were depolarized from a more negative holding potential (-90 mV); this finding was in contrast to observations in opossum esophageal body muscle (23). In a study of canine LES tissue strips (Salapatek et al., unpublished observations), as in this one, apamin had no significant effect, suggesting that unlike in canine intestine circular muscle (10, 24) apamin-sensitive, putative small KCa channels did not contribute to NO effects.

Outward currents through KCa channels were dependent on [Ca2+]pipette. When pipette Ca2+ was reduced to 8 nM, outward currents were reduced to levels similar to those with IbTX. Analysis of the relationship between [Ca2+]pipette and the magnitude of outward currents observed revealed an EC50 value of 108.5 ± 1.1 nM, which is similar to that for other Ca2+-dependent cNOS isoforms, such as human endothelial NOS with an EC50 for Ca2+-dependent activity of 100 nM [reviewed by Griffith and Stuehr (16)]. This finding suggested the possibility that varied [Ca2+]pipette affected cNOS rather than K Ca channels directly. Furthermore, the fact that the outward current through KCa channels could be reduced by the NOS inhibitor L-NNA (84.6 ± 0.8% reduction) despite high (1 µM) pipette Ca2+ levels implied that endogenous NO, from a continually active myogenic NOS, activated the outward current. The outward current activated by endogenous NO as well as that activated by exogenous NO (from NaNP) appears to be through KCa channels, since inhibition of their opening by IbTX precluded L-NNA effects and blocked NaNP effects. So far, we are unaware of evidence that IbTX affects K+ channels other than KCa channels. The specificity of the action of L-NNA actions for NOS was confirmed by the ability of either L-Arg or L-Cit (the NOS substrate acting at the same molecular site as L-NNA and the NOS product providing a substrate for regeneration of L-Arg, respectively) to prevent the L-NNA effect. Because L-Arg is charged whereas L-Cit is not, these findings make an interaction based on direct competition for K+ channels unlikely. These results support the hypothesis that an active myogenic NOS releases NO endogenously in an ongoing manner and activates an outward current similarly and continuously.

The reported [Ca2+]i levels required for activation vary with the transmembrane voltage, and the level of [Ca2+]i determines the activation at a given transmembrane voltage (2, 17, 25, 27). The local Ca2+ levels required for channel activation in smooth muscle vary widely for the various subtypes of KCa channels (from 10 nM to 100 µM). Activation is expected to occur whenever micromolar Ca2+ concentrations are present at the intracellular surface of the channel [reviewed by Kuriyama et al. (27)]. In our study, there was no significant difference in the peak outward current when pipettes contained 200, 1,000, or 8,000 nM Ca2+ or when NaNP was present and the pipette contained only 8 nM Ca2+. In this last case, the response to NaNP was shown to be blocked by IbTX and therefore to be due to current through KCa channels. Thus the Ca2+ level near the plasma membrane required for KCa activation under whole cell configuration experimental conditions was not determined by the concentration in the pipette or general cytoplasm. Van Breemen et al. (49) suggested that [Ca2+]i levels near the plasma membrane are controlled by "a superficial buffer barrier (SBB)" at levels higher than in the general cytosol due to vectorial release of Ca2+ from the SR toward the plasma membrane. Some hypothesis like the SBB is required to explain that the Ca2+ level near the plasma membrane was high enough to allow activation of KCa channels and maintain Ca2+ levels higher than deeper within the cell even when only 8 nM Ca2+ was present in the pipette.

The observation (see Table 1) that very high [Ca2+]i levels (8 µM) in LES cells did not activate KCa channels in the presence of L-NNA suggests that high [Ca2+]i alone may not activate these channels or that the [Ca2+]i levels near the plasmalemmal compartment where these channels are located can be lowered compared with cytosolic levels. Average [Ca2+]i levels in LES cells may normally be higher than in other smooth muscle cells to maintain sphincteric contraction or tone (reviewed in Ref. 11), but these levels may be differentially controlled near the plasmalemma to allow modulation of tone by activation of cNOS and KCa channels. This speculation warrants further study.

L-NNA as well as IbTX addition also caused significant smooth muscle cell depolarization under current-clamp conditions (+8.0 ± 0.3 mV) and cell contraction (whenever visual observations were made). Under these conditions, this cell membrane depolarization probably enhanced opening of voltage-dependent Ca2+ channels on the plasma membrane, resulting in Ca2+ influx and contraction. This possibility is consistent with our findings in LES muscle strips (43) in which blockade of myogenic NOS in the LES muscle strip resulted in contraction, whereas nifedipine, an L-type Ca2+ channel blocker, before or during L-NNA application, completely relaxed this active tension. A direct test of this in single cells has not been made because Ca2+ inward currents have not been observed in these cells even when K+ currents were abolished and Ba2+ was used as charge carrier (Salapatek and Daniel, unpublished observations). Single-channel studies are needed.

Nevertheless, nifedipine pretreatment resulted in a decrease in outward currents despite the presence of 1 µM Ca2+ in the pipette, and subsequent exogenous NaNP caused an increase in outward currents but not to previous control levels (56.2 ± 18.5% of control). Therefore, blockade of Ca2+ influx reduced NOS activation and endogenous NO production, in turn reducing KCa channel activation by endogenous NO. These results also imply that Ca2+ entrance through L-type Ca2+ channels contributes to the [Ca2+]i level near the plasmalemma, which permits KCa activation when exogenous NO is applied. Because exogenous NO (from NaNP) in the presence of nifedipine was unable to increase outward currents to control levels, NO cannot act simply to activate KCa channels irrespective of [Ca2+]i levels near the channel. Some investigators suggest that there is direct activation of KCa channels by NO (6), whereas others suggest that cGMP or PKG activates KCa channels directly or indirectly by increasing Ca2+ levels near the channel/plasma membrane by increasing Ca2+ influx and/or pumping into the SR (9, 30) and vectorial release of Ca2+ toward the plasmalemma via leak channels. However, to determine whether NO activation of KCa channels also involved manipulation of the L-type Ca2+ channel or of the SR Ca2+ pump, to affect [Ca2+]i levels near the plasmalemma to act directly on the channel or indirectly to enhance cNOS activity, further studies are needed.

Our results showed that when myogenic cNOS was maximally stimulated with high [Ca2+]i levels, no further recruitment of K Ca channels could occur with applied NO. However, when myogenic NOS was not maximally stimulated, KCa channels could be activated by exogenous NO whether pipette Ca2+ was 8 or 1,000 nM (Table 1). Presumably, under physiological conditions in vivo and in vitro in LES, there is submaximal activity of cNOS, since neural NO release causes an IJP and transient relaxation of LES, and NaNP also hyperpolarizes and relaxes the LES (22).

Murray et al. (30) reported that cystamine, a putative guanylate cyclase inhibitor, increased tone in opossum LES. This increase was tetrodotoxin insensitive. Thus a nonneural source of NO may exist in LES of another species and act through a guanylate cyclase-cGMP and/or PKG pathway. Application of cystamine in our experiments reduced outward currents 57.7 ± 2.9% from control levels. Therefore, in addition to a potentially direct action of NO on KCa channels, endogenous NO appears to act in part through a similar cGMP and/or PKG pathway.

These experiments and others on muscle strips (41, 43) suggested that an endogenous NOS exists in LES muscle cells that modulates LES smooth muscle excitability and contraction and that is activated continuously under physiological conditions. For cNOS to be continually activated, there must be, in addition to an appropriate [Ca2+]i level near the enzyme, a continuous supply of substrate (L-Arg). There was NADPH-diaphorase activity and membrane-bound cNOS activity in LES. The present results suggest that LES cells also contained AS and AL enzymes, which are capable of recycling L-Cit to L-Arg as suggested by our findings.

Our finding that L-Cit restored outward currents after L-NNA is the result that would be predicted if excess L-Cit resulted in increased regeneration of the NOS substrate, L-Arg. L-NNA and L-Arg compete at the same molecular site on NOS (7), and excess L-Arg displaces L-NNA, overcomes NOS inhibition, and restores NO production [as observed in our preparation of isolated cells and strips (43)]. Our result was similar to that of Shuttleworth et al. (45), who found that excess L-Cit (2 mM) overcame inhibition of nNOS by NG-nitro-L-arginine methyl ester (100 µM) or L-NNA (100 µM), restoring NO-mediated electric field-stimulated IJPs and effects on slow wave duration and frequency in the circular smooth muscle of the canine colon.

The AS antibody, given access to the cell from the patch-clamp pipette, gradually reduced outward currents. The simplest explanation is that the antibody prevented L-Arg regeneration and decreased NO production and its associated KCa channel activation. If L-Arg regeneration from L-Cit was blocked by the AS enzyme antibody, L-Arg application to the bath should still restore NO synthesis, as it did, but application of L-Cit should not and it did not. These observations support that immunoneutralization of AS enzyme activity blocks NO production by myogenic NOS due to insufficient NOS substrate (L-Arg) levels. They also suggest that continuous L-Arg regeneration supported NOS activity and NO synthesis in LES smooth muscle under our experimental conditions and could be supplied from L-Arg or from L-Cit, provided that AS activity was not blocked.

Alternate explanations of these findings are few and unlikely. For example, it might be argued that L-NNA directly inhibited the K+ channels sensitive to NO and that L-Arg somehow reversed this effect. There is no precedent for this possibility, and it is made even more unlikely by the fact that L-Cit is uncharged, in contrast to L-NNA or L-Arg, and also reverses the effect of L-NNA. Moreover, the ability of the antibody against AS to block outward currents in a time-dependent fashion and the block to be reversed by L-Arg but not L-Cit cannot readily be explained except by the dependence of continuous NO production on activity of the AS enzyme. There is no evidence to suggest that both the AS antibody and L-NNA have affinity for the K+ channel.

If L-Arg regeneration accounts for continuous activity of the cNOS in canine LES, then AS and AL enzymes must be constitutively present in LES smooth muscle. Our immunocytochemical findings support that possibility. They showed that antibodies against the two enzymes recognized LES muscle cells, as well as nNOS-containing neurons. This is not the first observation of constitutive expression of AS in smooth muscle. Hattori et al. (19) showed constitutive mRNA levels for the AL enzyme in murine aortic smooth muscle cell lines. Moreover, the AS and inducible NOS (iNOS) enzymes were coinduced by treatment with bacterial lipopolysaccharide (LPS) in combination with interferon-gamma . Nagasaki et al. (34) showed that iNOS AS and AL enzymes were coinduced in rats after treatment with LPS in vivo, prominently in the lung and spleen with one or the other enzyme induced to different extents in other tissues such as heart, liver, and testis. Yu et al. (51) showed that AS and AL enzymes are expressed in rat liver, kidney, and testis under basal, nonstimulated conditions. Also, like LPS-treated rats, AS or AL enzymes were expressed in other tissues of these animals to lesser extents. Therefore, they concluded that many tissues can act to convert L-Cit to L-Arg.

Hattori et al. (19) suggested that NOS AS and AL enzymes form a "channel cycle" and suggest that NOS would "preferentially use an arginine molecule recycled from citrulline (rather) than a new arginine molecule." This coinduction of the AS enzyme with iNOS allows vascular cells to overproduce NO during septic- and cytokine-induced circulatory shock. In this study, the coexpression of AS and AL enzymes constitutively appears to allow LES muscle to produce NO continually, implying an efficient reutilization of L-Cit.

We conclude that the weight of evidence supports that canine LES contains a continuously active cNOS that generates NO near the membrane, apparently dependent on Ca2+ entry through L-type Ca2+ channels, that operates to open K+ channels, modulate membrane potentials, and provide negative feedback to Ca2+ entry through voltage-dependent Ca2+ channels. Recycling of L-Cit to L-Arg contributes to maintenance of cNOS activity. The effects of NO on Ca2+ uptake by the SR and on properties of L-type Ca2+ channels still require study. The consequences of increased or decreased expression of this cNOS activity are so far unknown, as are any contributions of altered myogenic NOS activity to pathophysiological processes.

    ACKNOWLEDGEMENTS

We thank Dr. Ian Rodger of Merck-Frosst, Quebec, Canada, for his generous gift of the pharmacological agent, iberiotoxin.

    FOOTNOTES

This research was supported by the Medical Research Council of Canada.

Address for reprint requests: E. E. Daniel, Rm. 4N51, Health Sciences Centre, McMaster Univ., 1200 Main St. W., Hamilton, ON, Canada L8N 3Z5.

Received 14 January 1997; accepted in final form 12 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Allescher, H. D., I. Berezin, J. Jury, and E. E. Daniel. Characteristics of canine lower esophageal sphincter: a new electrophysiological tool. Am. J. Physiol. 255 (Gastrointest. Liver Physiol. 18): G441-G453, 1988[Abstract/Free Full Text].

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AJP Cell Physiol 274(4):C1145-C1157
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