Center for Swallowing and Motility Disorders, Brockton/West Roxbury Veterans Affairs Medical Center, West Roxbury 02132; and Harvard Medical School, Boston, Massachusetts 02125
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ABSTRACT |
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Nitric oxide (NO)
hyperpolarizes visceral smooth muscles. Using the patch-clamp
technique, we investigated the possibility that NO-mediated
hyperpolarization in the circular muscle of opossum esophagus results
from the suppression of a
Ca2+-stimulated
Cl current. Smooth muscle
cells were dissociated from the circular layer and bathed in
high-K+
Ca2+-EGTA-buffered solution.
Macroscopic ramp currents were recorded from cell-attached patches.
Contaminating K+-channel currents
were blocked with tetrapentylammonium chloride (200 µM) added to all
solutions. Raising bath Ca2+
concentration above 150 nM in the presence of A-23187 (10 µM) activated a leak current
(IL-Ca) with an
EC50 of 1.2 µM at
100 mV.
The reversal potential
(Erev) of
IL-Ca (
8.5 ± 1.8 mV, n = 8) was significantly
different (P < 0.05) from
Erev of the
background current (+4.2 ± 1.2 mV,
n = 8). Equimolar substitution of 135 mM Cl
in the pipette
solution with gluconate significantly shifted Erev of
IL-Ca to +16.6 ± 3.4 mV (n = 4)
(P < 0.05 compared with background),
whereas replacement of total Na+
with Tris+ suppressed
IL-Ca but did not
affect Erev
(
15 ± 3 mV, n = 3; P > 0.05).
IL-Ca was
inhibited by DIDS (500 µM). Diethylenetriamine-NO adduct (200 µM),
a NO
donor, and 8-bromo-cGMP (200 µM) suppressed IL-Ca by 59 ± 15% (n = 5) and 62 ± 21%
(n = 4) at
100 mV,
respectively. We conclude that in opossum esophageal smooth muscle
NO-mediated hyperpolarization may be produced by suppression of a
Ca2+-stimulated
Cl
-permeable conductance
via formation of cGMP.
calcium-activated current; reversal potential; nitric oxide donors; patch clamp; guanosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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NITRIC OXIDE (NO) hyperpolarizes many types of smooth
muscle and causes relaxation (14). The ionic mechanisms by which NO hyperpolarizes smooth muscle, however, are not fully understood. NO
donors activate multiple types of
K+ channel (12) and whole cell
K+ currents in smooth muscle that
are sensitive to either tetraethylammonium (TEA) (16), apamin and
quinine (9), or 4-aminopyridine (4-AP) (23). Moreover, in muscle
strips, the hyperpolarization elicited by NO donors, such as sodium
nitroprusside, 3-morpholinosydnonimine hydrochloride, or
S-nitrosothiols is partially inhibited
by apamin (3, 10), TEA and charybdotoxin (16), and quinine (3). Nitrergic inhibitory junction potentials (IJPs) in the opossum esophagus, however, are not blocked by TEA (up to 20 mM) (10), apamin
(3, 4, 6), glibenclamide (8), or 4-AP (3), suggesting that
these slow IJPs are not generated by the opening of
Ca2+-activated, ATP-sensitive, or
delayed rectifier K+ channels,
respectively. Quinine suppresses nitrergic IJPs (3) but only at
concentrations that block cation and
Cl channels (7).
In previous studies in opossum esophageal smooth muscle (4) and in the
guinea pig ileum (5), we reported that the nitrergic slow IJP is caused
by the suppression of a resting
Cl conductance. This
conclusion was based on the effects of
Cl
substitution and
putative Cl
channel
blockers on the resting membrane potential and on slow IJPs in muscle
strips from these tissues (4, 5). Moreover, a
Ca2+-activated
Cl
current in the circular
muscle cells of the opossum esophagus has recently been characterized
(22). The purpose of the present study was to identify
Cl
channel current in
esophageal circular smooth muscle cells using cell-attached patch-clamp
recordings and to examine the effect of a NO donor,
diethyenetriamine-NO (DETA-NO) (15), and cGMP on these currents. These
studies reveal that DETA-NO and 8-bromo-cGMP (8-BrcGMP) both suppress a
Ca2+-stimulated
Cl
current and provide
strong support for the view that nitrergic IJPs may be mediated by
closure of a Cl
conductance.
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METHODS |
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Isolation of esophageal smooth muscle cells. Opossums were killed by lethal injection of pentobarbital sodium (40 mg/kg ip) in accordance with guidelines of the Animal Studies Committee, West Roxbury Veterans Affairs Medical Center. After a midline incision below the sternum, the lower esophagus was removed and placed in modified Hanks' solution containing 10 µM added Ca2+. Single smooth muscle cells were prepared as described previously, using collagenase and trypsin digestion of tissue pieces (22).
Patch-clamp recordings.
Aliquots of smooth muscle cells were placed in the cell chamber on an
inverted microscope (Olympus) and allowed to adhere to the glass
surface. The cell chamber was then perfused continuously (0.5 ml/min)
with high-K+ physiological
solution of the following composition (in mM): 150 KCl, 1 MgCl2, 10 HEPES, 5 D-glucose, 1 CaCl2, and 1.38 EGTA. The pH was
adjusted to 7.2 with 10 M KOH. The
Ca2+ concentration
([Ca2+]) of this
solution was calculated to be ~150 nM using a computer program and
known binding constants between EGTA and
Ca2+ (Eqcal; Biosoft). Patch
pipettes were drawn from borosilicate capillary glass (Kimax 51 no.
34502; Fisher) on a programmable puller (Sutter P80; Novato) and fire
polished (Narishige) to have resistances of 5-10 M when filled
with the standard pipette-filling solution of the following composition
(in mM): 150 NaCl, 2.5 KCl, 10 HEPES, 5 D-glucose, and 2 MgCl2. The pH of this solution was adjusted to 7.2 with NaOH. The
Ca2+-stimulating solution (CSS)
consisted of high-K+ solution
containing 1 µM Ca2+ and 10 µM
A-23187. Ramp voltages (+50 to
100 mV, over 4 s) were delivered,
and currents were recorded using an Axopatch 200A amplifier (Axon
Instruments) and digitized using a Labmaster analog-to-digital converter coupled to a Pentium PC running pClamp 6.02 software (Axon
Instruments). Currents were filtered at 1 kHz, and data were analyzed
using pClamp software. Liquid junction potentials were canceled prior
to seal formation. Junction potentials between the pipette and bathing
solutions were less than 5 mV (as measured separately using a 3 M KCl
agar bridge), and the reveral potentials (Erev) have not
been corrected for these values. Prior to averaging, ramp currents were
corrected for a linear leak current, which was recorded from
cell-attached patches in low-Ca2+
(150 nM) bathing solutions. These
Ca2+-insensitive currents were
assumed to represent nonionic current flow across the seal resistance.
All recordings were obtained at room temperature
(22-24°C).
Drugs.
DETA-NO (Research Biochemicals), which has a half-life of >20 h at pH
7.4 (15), was dissolved directly in the perfusate. 8-BrcGMP (Sigma) was
also dissolved in the perfusate, and tetrapentylammonium chloride (TPA;
Aldrich or Sigma) was made up as an aqueous stock solution
(101 M). Stock solutions of
A-23187 (10
2 M; Molecular
Probes), DIDS (10
1 M;
Sigma), and LY-83583 (2 × 10
1 M; Calbiochem) were
dissolved in pure DMSO.
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RESULTS |
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Ca2+-stimulated
currents in esophageal circular smooth muscle cells.
Smooth muscle cells were perfused initially with a
high-K+,
low-Ca2+ (150 nM) physiological
solution (PS) to null the resting potential. Currents were recorded
using pipettes filled with low-K+
(2.5 mM) solution from cell-attached patches in response to ramp hyperpolarizations (+50 to 100 mV over 4 s) from a holding
potential of 0 mV. Fifteen consecutive ramp currents, generated at 5-s
intervals, were digitally averaged to obtain a mean ramp current for
analysis. Contamination from K+
channel currents was minimized by adding TPA (200 µM) (2) to all the
solutions. Under these conditions, the background current reversed at
+4.2 ± 1.2 mV (n = 8) (see Fig.
2A).
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Effect of Cl and
Na+
substitution.
The presence of fixed intracellular negative charges is expected to
result in a lower intracellular than extracellular
[Cl
],
establishing a negative Cl
Nernst potential
(ECl). Because
the Erev of
IL-Ca lies
between the K+ equilibrium potential
(EK; ~100 mV)
and 0 mV, the ionic nature of
IL-Ca may be
anionic or of a mixture of anions and cations. To test whether
IL-Ca is carried
by Cl
, we reduced the
[Cl
] in the
pipette solution to 21.5 mM by equimolar replacement with gluconate.
Under these conditions, the
Erev of the
background current recorded in 150 nM
Ca2+ (+7 ± 4 mV,
n = 4) did not differ significantly
(P > 0.05) from the corresponding
background current recorded under a normal
Cl
gradient (+4.2 ± 1.7 mV, n = 8). In the presence of reduced
extracellular [Cl
], however,
the Erev of the
Ca2+ (1 µM)-stimulated
IL-Ca was shifted
significantly positive to +16.6 ± 3.4 mV
(n = 4)
(P < 0.05) (Fig.
3, traces
i and iii). The
relative shift in
Erev of
IL-Ca was
approximately one-half the value predicted for a
Cl
current from the Nernst
equation. These data suggest that although IL-Ca is carried
largely by Cl
, cations such
as Na+ and
K+ may also contribute to this
current and account for the discrepancy between the predicted and
actual changes in
Erev of
IL-Ca.
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Effect of DETA-NO on
IL-Ca.
To investigate whether NO can inhibit
IL-Ca, we tested
the effect of DETA-NO, a stable NO · donor (8, 15). After
activation of
IL-Ca, DETA-NO
(200 µM) was perfused continuously through the cell chamber,
resulting in a marked decrease in the slope of ramp currents (Fig.
4A).
IL-Ca recovered
upon washout of DETA-NO. In five cells tested,
IL-Ca was
decreased by 50 ± 11% at +50 mV, 65 ± 16% at 50 mV,
and 59 ± 15% at
100 mV. In the cell depicted in Fig.
4A, this inhibition was prevented by
pretreatment with LY-83583 (200 µM), an inhibitor of soluble
guanylate cyclase (19), suggesting that the action of NO is mediated by
cGMP.
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DISCUSSION |
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In the present study we have demonstrated that smooth muscle cells of
the opossum esophageal circular muscle express a
Ca2+-stimulated
Cl conductance that is
suppressed by NO via stimulation of guanylate cyclase. Replacement of
extracellular Na+ with
Tris+ failed to shift
Erev of this
current significantly. Substitution of extracellular
Cl
with gluconate, shifted
the Erev of
ICl-Ca
positively. Although this shift was approximately half the value
expected for a purely Cl
-selective conductance,
this is consistent with the known poor selectivity of many
Cl
channels, including
small-conductance Ca2+-activated
Cl
channels in bovine
pulmonary artery endothelial cells (18, 22).
In cell-attached patches,
ICl-Ca was
stimulated by levels of Ca2+ that
are achieved in intact tissue. Channel activity, however, had a
tendency to decrease after patch excision despite the presence of the
same high level of Ca2+ on the
cytoplasmic surface of patches, indicating that soluble intracellular
mediators may be involved in activation of
ICl-Ca by
Ca2+. This phenomenon has been
reported previously for large-conductance Cl channels in chicken
myotubes (20) and also for small-conductance Ca2+-activated
Cl
channels in vascular
smooth muscle (11). In our recordings, ramp currents consisted mainly
of poorly resolvable openings of small-conductance channels. However,
channels with apparently large (~300 pS) conductances were also
stimulated with high Ca2+ (>1
µM) in the same patches with less frequency. Sun et al. (21) have
previously described "maxi"
Cl
channels in inside-out
patches from rabbit colonic smooth muscle cells. The large-conductance
Cl
channels were
insensitive to cytoplasmic
[Ca2+] up to 0.5 mM
and may represent a different population of
Cl
channel (see Ref. 13).
Further studies are required to elucidate the single channel properties
of ICl-Ca in the
opossum esophagus.
An important finding in our present study has been that the
ICl-Ca is
suppressed by the NO · donor DETA-NO (15), and this effect is
antagonized by LY-83583, a blocker of cytosolic guanylate cyclase (19).
This suggests that NO acts to suppress
ICl-Ca via
intracellular accumulation of cGMP. This conclusion is further supported by our observation that 8-BrcGMP, a cell-permeable analog of
cGMP, also suppressed this
Cl current. Although NO has
been shown to suppress L-type
Ca2+-channel currents in
esophageal cells (1), which may indirectly lead to suppression of
ICl-Ca,
intracellular [Ca2+]
in our cells was most likely clamped close to 1 µM with
Ca2+ ionophore. The suppression of
ICl-Ca by DETA-NO
therefore cannot be explained by inhibition of
Ca2+-channel currents. Although
the mechanism by which cGMP inhibits ICl-Ca was not
addressed in the present study, inhibition of a similar
Ca2+-activated current in mouse
ileal myocytes by DETA-NO is blocked by pretreatment with H-7, a
nonspecific kinase inhibitor (F. Vogalis and R. K. Goyal, unpublished
observations). This suggests that cGMP is not the final mediator in the
suppression of
ICl-Ca.
It has been shown recently that in rat cerebral arteries,
Cl channels are responsible
for the maintenance of membrane potential and myogenic tone (17). In
intact esophageal muscle strips, a resting
Cl
conductance has been
previously shown to maintain the membrane potential positive of
EK (5). It is
therefore possible that tonic stimulation of
ICl-Ca by ongoing
Ca2+ trafficking between the cell
membrane and stores at physiological temperatures may contribute to the
depolarized resting potential in intact esophageal smooth muscle.
Suppression of this current by neurally released NO would then allow
the membrane to be hyperpolarized by a resting
K+ conductance, giving rise to
slow IJPs that are recorded in the esophageal smooth muscle and in
other visceral smooth muscle preparations. Consistent with this
hypothesis are the observations that nitrergic IJP in the opossum
esophagus is associated with an increase in membrane resistance and is
inhibited by DIDS (4), a blocker of
Cl
channels (13). In
contrast to the opossum esophagus, nerve stimulation in the circular
muscle of the guinea pig ileum and the mouse stomach evokes purinergic
fast IJPs as well as nitrergic slow IJPs. Fast IJPs are known to be
generated by the opening of apamin-sensitive
K+ channels, whereas slow IJPs may
be generated by suppression of a resting
Cl
conductance (5, 8).
In summary, we have demonstrated that DETA-NO, a NO donor, inhibits a
Ca2+-stimulated leak current
carried by Cl. Inhibition
of this current may be an important mechanism by which NO released from
nitrergic motor nerves produces smooth muscle hyperpolarization and
inhibits electromechanical coupling.
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ACKNOWLEDGEMENTS |
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This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-31092 (to R. K. Goyal) and DK-50137 (to F. Vogalis).
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FOOTNOTES |
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Portions of this study have been published previously in abstract form (J. Gen. Physiol. 110: 37A, 1997).
Address for reprint requests: R. K. Goyal, Research & Development 151, Brockton/West Roxbury VA Medical Center, 1400 VFW Parkway, West Roxbury, MA 02132.
Received 11 August 1997; accepted in final form 24 January 1998.
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