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
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ABSTRACT |
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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,
104 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
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INTRODUCTION |
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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(41, 43), and we obtained preliminary evidence for a myogenic NOS in other sphincters along the gastrointestinal tract (unpublished observations).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).
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METHODS |
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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
M. 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.
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 ![]() |
RESULTS |
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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
105 M carbachol, returning
to their previous elongated shape when these agents were removed. Only
cells with an access resistance of <25 M
were studied. In these
experiments, access resistance ranged from 8 to 21 M
, 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
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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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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 (106 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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Endogenous NOS Inhibition
When the NOS inhibitor L-NNA (10
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The specificity of L-NNA for NOS
was confirmed by the ability of
L-Arg
(103 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
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When endogenous NOS activity was blocked with
L-NNA
(104 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 × 107 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
(103 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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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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DISCUSSION |
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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-. 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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ian Rodger of Merck-Frosst, Quebec, Canada, for his generous gift of the pharmacological agent, iberiotoxin.
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FOOTNOTES |
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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.
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