Nitric oxide suppresses a Ca2+-stimulated Clminus current in smooth muscle cells of opossum esophagus

Yong Zhang, Fivos Vogalis, and Raj K. Goyal

Center for Swallowing and Motility Disorders, Brockton/West Roxbury Veterans Affairs Medical Center, West Roxbury 02132; and Harvard Medical School, Boston, Massachusetts 02125

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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 NObullet 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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 MOmega 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 (10-1 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.

Tests of statistical significance (P < 0.05) were performed between means using Student's t-test, where n represents the number of cells.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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).

Cells were then perfused with CSS containing 1 µM Ca2+ and 10 µM A-23187. This increased the slope of the ramp currents, which reached a maximum value within 10-15 min. The Erev of the Ca2+-stimulated "leak" current (IL-Ca), which averaged -8.5 ± 1.8 mV (n = 8), differed significantly (P < 0.05) from that of the background current (Fig. 1). Patch excision usually resulted in rundown of this current. Therefore the sensitivity of IL-Ca to Ca2+ was studied in cell-attached patches by varying the [Ca2+] in the high-K+ PS, in the continuous presence of Ca2+ ionophore (A-23187; 10 µM). Increasing the [Ca2+] in the bathing solution caused an increase in the magnitude of IL-Ca throughout the voltage ramp (Fig. 2A). To estimate the Ca2+ dependence of IL-Ca, the mean currents measured at -100, -50, and +50 mV were fitted with a Hill function of unitary slope. This yielded EC50 values of 1.2, 0.6, and 1.6 µM, respectively (Fig. 2B), suggesting that the leak channels stimulated by Ca2+ are not appreciably voltage dependent.


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Fig. 1.   Activation of an inward "leak" current by Ca2+-stimulating solution (CSS), a high-K+ solution containing 1 µM Ca2+ and 10 µM A-23187, in a smooth muscle cell of opossum esophagus. Shown are ramp currents averaged from 15 consecutive traces at various times after application of CSS recorded from same cell. Current achieved maximal amplitude at 12 min. Note that current was linear between -50 and +50 mV but showed some rectification at more negative potentials. Cell was bathed in high-K+ physiological solution containing 150 nM Ca2+ and tetrapentylammonium chloride (200 µM) to block K+ channels. Ionic current was recorded from a cell-attached patch in response to ramp hyperpolarizations from +50 mV to -100 mV, over 4 s.


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Fig. 2.   Ca2+ sensitivity of Ca2+-stimulated leak current (IL-Ca). A: ramp currents averaged from 15 consecutive traces recorded after equilibration at different levels of Ca2+ and A-23187 (10 µM) from the same cell. Note that amplitude of currents increased with increasing Ca2+ concentration in the bathing solution. Currents were recorded from the same cell-attached patch in response to ramp hyperpolarizations in the presence of CSS. B: normalized amplitude of averaged ramp currents measured at +50 mV (open circle ), -50 mV (square ), and -100 mV (triangle ) plotted as a function of [Ca2+] in the CSS. Data points are means ± SE of 4 patches (i.e., n = 4 cells) and were fitted with the Hill equation, which yielded EC50 values of 1.6, 0.6, and 1.2 µM at +50, -50, and -100 mV, respectively, with the slope fixed at unity. Data indicate that Ca2+-stimulated current is activated by physiological levels of Ca2+ and is not appreciably voltage dependent.

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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Fig. 3.   Representative averaged ramp currents recorded from cell-attached patches showing effect of external Cl- and Na+ substitution on IL-Ca. Reduction of external (pipette) Cl- concentration to 21.5 mM by equimolar replacement with gluconate shifted the reversal potential (Erev) of IL-Ca ~30 mV positive from -14 mV (trace i) to +16 mV (trace iii). Substitution of Na+ with equimolar (150 mM) Tris+ suppressed the amplitude of IL-Ca but failed to shift Erev (trace ii), suggesting that IL-Ca is carried by Cl-. Ramp currents were recorded in CSS from 3 different patches (cells), and the averaged current from 15 traces from each patch is plotted as a function of ramp potential.

To test the possibility that IL-Ca may be carried by Na+, we fully substituted NaCl in the pipette solution with Tris · Cl, and in three cells tested, Erev of IL-Ca was not significantly altered (Tris+: -15 ± 3 mV, n = 3; control: -8.5 ± 1.8 mV, n = 8; P > 0.05) (Fig. 3, trace ii). This suggests that under normal conditions IL-Ca is not carried by cations such as Na+ and is mainly generated by Cl-. Moreover, DIDS (500 µM), a blocker of Cl- channels, suppressed IL-Ca in esophageal cells (n = 3). The decrease in the amplitude of IL-Ca after Na+ substitution suggests a regulatory effect of Na+ on this current. Together, these observations indicate that IL-Ca is carried predominantly by Cl-.

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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Fig. 4.   Suppression of IL-Ca by diethylenetriamine-nitric oxide adduct (DETA-NO) and cGMP. A: suppression of inward IL-Ca (control) by DETA-NO (200 µM) and inhibition of this effect by concomitant application of LY-83583 (200 µM) and DETA-NO. Ramp currents are averages of 15 consecutive traces recorded under different conditions from a cell-attached patch. B: suppression of IL-Ca by 8-bromo-cGMP (8-BrcGMP; 200 µM), in a different cell. Ramp currents were generated as in A. Addition of 8-BrcGMP (200 µM) to the bathing solution inhibited IL-Ca. Pretreatment with LY-83583 (200 µM), an inhibitor of guanylate cyclase, failed to prevent suppression of this current by concomitant addition of 8-BrcGMP.

To confirm that cGMP inhibits IL-Ca, we tested the action of membrane-permeable 8-BrcGMP on IL-Ca as shown in Fig. 4B. Bath perfusion of 8-BrcGMP (200 µM) rapidly suppressed IL-Ca, by an average of 62 ± 17% at +50 mV, 64 ± 20% at -50 mV, and 62 ± 21% at -100 mV, in four cells tested. Washout of 8-BrcGMP led to the recovery of IL-Ca. However, the inhibitory action of 8-BrcGMP was unaffected by pretreatment with LY-83583 (200 µM). These data suggest that the inhibitory actions of NO are likely to be mediated by cGMP.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

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).

    FOOTNOTES

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.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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