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J Thorac Cardiovasc Surg 1995;110:157-164
© 1995 Mosby, Inc.

GENERAL THORACIC SURGERY

Role of nitric oxide in human esophageal circular smooth muscle in vitro

Boston Mass. Durham N.C.

This work was supported by National Institutes of Health grant DK 34854 (Harvard Digestive Disease Center). J.S. is a Pew scholar in the Biomedical Sciences and the recipient of a Clinical Investigator Development award from the National Institutes of Health (HL02582).

Received for publication May 26, 1994. Accepted for publication Nov. 1, 1994. Address for reprints: David J. Sugarbaker, MD, Division of Thoracic Surgery, Brigham & Women's Hospital, 75 Francis St., Boston, MA 02115.

Abstract

The role of nitric oxide in human esophageal smooth muscle was examined. Immunostaining for constitutive nitric oxide synthase labeled nerve fibers and bundles within longitudinal and circular smooth muscle layers of resected tissue samples. Strips of circular muscle mounted in organ baths exhibited spontaneous contractions and active tone. When exposed to 5-second trains of electric field stimulation at 20 Hz, most strips exhibited intrastimulus "on" and poststimulus "off" contractions. Exposure to a 0.1µmol/L (or greater) concentration of atropine converted "on" contractions to "on" relaxations and reduced "off" contractions by 63%. Exposure to N^G-nitro-L-arginine resulted in concentration-dependent enhancement of "on" contractions and abolition of "off" contractions. Excess L-arginine enhanced the reversal of these effects. Sodium nitroprusside inhibited both spontaneous and evoked contractions. These results suggest that nitric oxide synthesis is a mediator of neural inhibition of human esophageal circular smooth muscle and is necessary for the occurrence of "off" contractions. (J THORACCARDIOVASCSURG1995;110:157-64)

Peristalsis in the distal segment of the human esophagus is poorly understood. Experiments with animals in vivo have revealed that esophageal peristalsis involves coordinated contraction of the inner muscularis mucosa and the outer longitudinal and circular layers of smooth muscle. ¹ Contractions that resemble peristalsis survive bilateral vagotomy ² and occur in vitro, ³ suggesting that mechanisms capable of controlling sequential contraction exist within the intramural nerve plexuses. In the opossum, the circular muscle is initially hyperpolarized during stimulation or swallow induction and subsequently depolarizes, fires action potentials, and contracts as the peristaltic wave passes the recorded region. ^{1, 2} Electric field stimulation (EFS) in vitro produces intrastimulus "on" and poststimulus "off" contractions in strips of animal ^4-6 and human ⁷ circular muscle. The "on" response is likely to represent EFS-produced activation of cholinergic nerves, as it is abolished by atropine. ⁶ The "off" contraction is apparently related to EFS-produced nonadrenergic, noncholinergic neural inhibition and may represent a "rebound" effect. ⁸

Nitric oxide has been proposed as a mediator of nonadrenergic, noncholinergic inhibition of esophageal smooth muscle based on the results of animal studies. ^9-12 In this study, localization of constitutive nitric oxide synthase (c-NOS) within the human esophageal muscularis externa was accomplished by immunostaining, and the role of nitric oxide synthesis in the in vitro field-stimulated responses of human circular muscle was investigated with the use of the competitive inhibitor N^G-nitro-L-arginine (L-NNA). Preliminary reports of some of these experiments have been published. ^{13, 14}

MATERIALS AND METHODS

Tissue procurement.
Samples of smooth muscle were obtained from uninvolved regions (7 to 11 cm above the lower esophageal sphincter) of surgical specimens (n = 7) resected for esophageal cancer. A protocol for experimental use of human discarded material was approved by the Committee for the Protection of Human Subjects from Research Risk at Brigham and Women's Hospital. Samples were placed in Dulbecco's modified Eagle's medium gassed with 95% oxygen and 5% carbon dioxide on ice within minutes of resection.

Immunohistochemistry.
The presence of c-NOS in this tissue was determined by immunostaining. Cryostat sections (8 µm) obtained from O.C.T. (Tissue-Tek, Miles, Inc., Elkhart, Ind.) embedded slices of esophageal tissue consisting of both longitudinal and circular muscle were exposed to a polyclonal rabbit antibody preparation that was generated by immunization with rat brain c-NOS and that had undergone affinity purification on c-NOS antigen. ¹⁵ The sections were first incubated overnight with primary specific c-NOS antibody or, in the case of controls, with dilutions of commercially prepared rabbit immunoglobulin G. A biotin-conjugated goat antirabbit immunoglobulin G, avidin-biotin peroxidase complex (Vector Labs, Burlingame, Calif.) and a substrate solution were used to detect specific binding. The substrate solution consisted of 0.03% hydrogen peroxide and a 2 mg/ml concentration of diaminobenzidine in Tris-saline solution (0.5 mol/L) with imidazole (1 mol/L) and 0.3% azide (to block endogenous peroxidase) at pH 7.7. Sections were counterstained lightly with hematoxalin, dehydrated, and covered with a coverslip.

Organ bath experiments.
Each sample was pinned with the mucosal surface up in a Sylgard-bottom Petri dish (Dow Corning Corp., Midland, Mich.) and immersed in cold, gassed Dulbecco's medium. The mucosa was removed by sharp dissection. Strips of circular muscle were cut parallel to the fibers and dissected free from the longitudinal muscle layer with a dissecting microscope. Each strip was approximately 1 cm by 3 mm and contained only circular fibers.

The strips were suspended in water-jacketed organ baths, which accommodated two strips each. One end of each strip was anchored, and the other end was attached with stainless steel hooks to a force transducer (World Precision Instruments, Sarasota, Fla., FORT-10) for isometric tension recording. Tension signals from the force transducers were amplified (World Precision, TBM-4) and digitized (Axon Instruments, Foster City, Calif., TL-1, Axotape) at 10 Hz on-line. Each strip was positioned between two platinum stimulating electrodes (5 mm apart). Strips were immersed in 50 ml continuously gassed Krebs buffer (composition, in millimoles per liter: Na⁺, 138.6; K⁺, 5.9; Ca⁺⁺, 2.5; Mg⁺⁺, 1.2; Cl^- , 134; HCO₃^- , 15.5; H₂PO₄^- , 1.2; glucose, 11.5; pH 7.4 ± 0.05) at 37º ± 0.5º C. All bathing solutions used during the experiments consisted of freshly prepared, prewarmed, pregassed buffer with or without additional dissolved substances. All reagents were obtained from Sigma Chemical Company (St. Louis, Mo.) unless otherwise stated. After 5 minutes the strips were gradually stretched to 130% of their original length. This degree of stretch was determined in pilot studies (two specimens) to result in optimal responses to EFS. All strips were allowed to equilibrate 1 hour before stimulation.

EFS was administered via the platinum electrodes that were connected to an isolated stimulator (Grass Instrument Co, Quincy, Mass., SIU5, S11) programmed to deliver trains of square pulses with parameters (120 V; 20 Hz; 1 msec pulse duration; 5-second train duration) producing optimal responses in this tissue, based on pilot studies in two specimens and in agreement with published data. ⁷ EFS was administered to each strip twice or four times per series at 2-minute intervals, and responses were averaged over the series. Series of EFS were repeated until responses to EFS exhibited stable amplitudes. The response to the final EFS series in buffer alone was considered the control response. Subsequent "off" responses, recorded during incubation in L-NNA, were expressed as a percentage of control. Because control "on" responses often had zero or negative amplitudes, "on" contractions in L-NNA were expressed as a percentage of the maximum (asymptotic) "on" response recorded.

So that the effects of nitric oxide synthesis on EFS-produced responses could be determined, solutions of Krebs buffer containing L-NNA were added to each bath in 50 µl volumes to produce a series of cumulatively increasing concentrations (10 nmol/L to 300 µmol/L), and EFS (20 Hz) was administered after 5 minutes' incubation at each concentration. After exposure to L-NNA, each organ bath was rinsed twice with 50 ml fresh buffer. To determine whether excess L-arginine would lead to recovery of "off" responses after L-NNA exposure, we arbitrarily divided the strips into two groups. L-arginine (10 µmol/L final concentration) was then added to the organ baths of one group, whereas the other group remained in Krebs buffer and served as controls. Series of EFS were applied at 10-minute intervals to all strips. In a separate group of strips (n = 8), sodium nitroprusside was added in 50 µl of buffer (final concentration 1 to 30 µmol/L) to assess the responses of strips to exogenous nitric oxide.

Statistics.
Computer-assisted analysis of tension recordings was accomplished with custom software. Digitized waveforms were plotted, allowing on-screen measurement of various parameters (e.g., average tone, response amplitude, latency) with adjustable cursors. Median effective concentrations (EC₅₀s) were calculated for L-NNA by linear substitution after regression of the steep, approximately linear portion (3 to 30 µmol/L concentration of L-NNA) of the concentration/response plots (which encompassed the 50% point in each case). Statistical treatment involved repeated-measures analysis of variance and matched-pairs or two-group t tests using a commercial software package (BMDP Statistical Software, Inc., Los Angeles, Calif.). Values are expressed as mean ± standard error. For analyses of variance, F ratios associated with main effects and interactions are reported with parenthetical (between, within) degrees of freedom and tail probabilities.

RESULTS

Immunostaining for c-NOS resulted in dense labeling of nerve bundles in the connective tissue between circular and longitudinal muscle layers and of nerve fibers among the smooth muscle fibers of both layers (Fig. 1, A). No structures were labeled by control antibody (Fig. 1, B), demonstrating the specificity of localization of the c-NOS antigen.

View larger version (271K): [in this window] [in a new window]   Fig. 1. A,Immunoperoxidase staining of human esophageal smooth muscle demonstrating selective labeling of c-NOS in nerve bundles and fibers within the circular (CM) and longitudinal (LM) layers. B, These structures were not labeled by control antibody. Calibration: 50 µm.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Isometric tension recordings from a single human circular smooth muscle strip illustrating the effects of L-NNA and L-arginine on the response to electric field stimulation. Each panel depicts the response to a single train, the onset and offset of which are indicated by vertical ticks. A, Control responses exhibited by this strip in Krebs buffer consisted of a relaxation, followed by a poststimulus "off " contraction. Spontaneous phasic contractions are visible before the onset of stimulation. B to D, Exposure to increasing concentrations of L-NNA resulted in the disappearance of the "on" relaxation, the emergence and concentration-dependent enhancement of a stimulation-induced "on " contraction, and a concentration-dependent diminution of the "off " contraction. E, Sixty minutes' incubation in L-arginine after washout of L-NNA resulted in decrement of the "on " contraction and recovery of the "off " contraction. Calibration: 1 gm x 5 seconds.

Cumulative addition of L-NNA (n = 14) resulted in a concentration-dependent (F[9,117] = 532; p < 0.0001; EC₅₀ = 7.9 µmol/L) enhancement of "on" contractions (Fig. 2, B to D, and Fig. 3, A), which generally became monophasic in higher concentrations of L-NNA. The L-NNA-enhanced "on" contraction was reduced in amplitude by 92% ± 3% after the addition of atropine (final concentration 1 µmol/L) and then abolished after the further addition of hexamethonium (final concentration 10 µmol/L). Repeated-measures analysis of variance revealed a time-dependent reversal of these effects of L-NNA (F[6,72] = 105; p < 0.0001) after washout of the antagonist. This reversal was significantly augmented by the presence of excess L-arginine (Figs. 2, E, and 3, B), as revealed by a significant main effect of L-arginine exposure (F[1,12] = 13.79; p < 0.005) and a significant interaction between L-arginine exposure and time variables (F[6,72] = 34.07; p < 0.0001).

View larger version (34K): [in this window] [in a new window]   Fig. 3. Average intrastimulus "on" contractions exhibited by human circular smooth muscle strips in response to EFS during and after exposure to cumulatively increasing concentrations of L-NNA. Area under the response waveform is plotted as a percentage of the largest response. Error bars are ±1 standard error. A, Exposure to L-NNA resulted in concentration-dependent enhancement of "on" contractions. B, Time-dependent diminution of enhanced "on" contractions after washout of L-NNA was significantly enhanced in the group of strips incubated in L-arginine (solid line) as compared with the Krebs control group (dashed line).

View larger version (38K): [in this window] [in a new window]   Fig. 4. Average poststimulus "off " contractions exhibited by human circular smooth muscle strips in response to EFS during and after exposure to cumulatively increasing concentrations of L-NNA. Peak "off " contraction amplitude is plotted as a percentage of the pre-L-NNA control amplitude. Error bars are ±1 standard error. A, Exposure to L-NNA resulted in concentration-dependent diminution and eventual abolition of "off " contractions. B, Strips exposed to L-arginine (solid line) exhibited a time-dependent recovery of almost 90% of pre-L-NNA control response amplitude, whereas control strips not exposed to L-arginine (dashed line) exhibited only minimal recovery of "off" responses.

The results of this study indicate that (1) c-NOS immunoreactivity is characteristic of nerves and nerve fibers in human esophageal smooth muscle and (2) inhibition of nitric oxide synthesis with L-NNA results in reversible and concentration-dependent alteration of contractile responses elicited by EFS (i.e., enhancement of "on" contractions and diminution of "off " contractions).

These results are consistent with a two-component interpretation of the response of human esophageal circular muscle strips to EFS, and they imply a two-component neuromuscular mechanism for peristalsis in the smooth muscle esophageal segment. In vitro, both excitatory and inhibitory components are activated during a stimulus train, producing mechanical relaxation, contraction, or mixed responses. This inconsistency is probably an artifact resulting from the nature of EFS, which apparently activates multiple intramural nerves simultaneously. Under most circumstances, only the excitatory component is evident during poststimulus responses. The inhibitory component is diminished by inhibitors of nitric oxide synthesis, implicating nitric oxide as a mediator. This interpretation is supported by finding that nerves and nerve fibers stain densely for c-NOS. The excitatory component consists of a cholinergic subcomponent, which predominates during intrastimulus responses and accounts for at least 63% of the poststimulus contraction, and a noncholinergic subcomponent, which is absent or inhibited during stimulation and which is manifest during the "off" response.

Studies of esophageal circular muscle in several species have provided evidence of a nonadrenergic, noncholinergic inhibitory innervation of this tissue. Inhibitory junction potentials have been recorded during EFS in vitro from opossum ¹⁶ and cat ¹⁷ circular muscle strips and during swallowing from exposed opossum circular muscle in vivo. ¹ Evidence of an inhibitory response in human circular muscle was observed in the present study in the form of intrastimulus relaxation of strips not initially exhibiting "on" contractions and of those exposed to atropine. Cholinergic "on" contractions were augmented by exposure of strips to L-NNA, suggesting that this agent interfered with the inhibitory component of the response and that nitric oxide synthesis contributes to nonadrenergic, noncholinergic inhibition. This thesis is supported by the observation that sodium nitroprusside, which releases nitric oxide into solution, reversibly relaxed circular muscle strips and abolished both spontaneous and evoked contractions. Nitric oxide synthesis has also been found to mediate relaxation of smooth circular muscle of the human lower esophageal sphincter. ^{18, 19}

The inhibitory component of the response to EFS has been assumed to be related to the occurrence and timing of the "off " contraction. The in vitro "off " contraction is analogous to the peristaltic circular muscle contraction, in that both contractions occur after a latent period associated with neurogenic inhibition. One hypothesis holds that the "off " contraction occurs as a result of a rebound effect as the smooth muscle membrane is released from EFS-produced inhibition and that the duration of the inhibitory junction potential controls the latency of the contraction. ⁵ Because this latency is regionally graded, the inhibitory component may have a role in controlling sequential peristaltic contraction in the intact organ. ⁵ In the present study, L-NNA produced a concentration-dependent diminution of the "off " contraction, which indicates a role of nitric oxide synthesis in a critical component of this response. The effect of L-NNA on the "off" contraction mirrored its antagonism of the inhibitory component (as indicated by "on" contraction enhancement), a result that is not inconsistent with a "rebound" hypothesis.

Esophageal manometry in human subjects has revealed that cholinergic manipulations profoundly affect peristaltic contractions during swallowing. Intramuscular ²⁰ or intravenous ²¹ atropine reduced the incidence of swallow-associated peristalsis, as well as the amplitude and velocity of peristaltic contractions in healthy subjects. The cholinesterase inhibitor edrophonium increased the amplitude and duration of peristaltic contractions and decreased the peristaltic velocity. ²² Correspondingly, the results of the present experiment revealed a conspicuous contribution of cholinergic excitation to both "on" and "off " responses of human esophageal circular muscle to EFS. The "on" contraction was abolished by atropine alone, as has been reported in the opossum. ⁶ However, when the inhibitory component of the EFS response was antagonized by L-NNA, the enhanced "on" contraction was only 92% reduced by atropine. The residual contraction was abolished by the further addition of hexamethonium. The sensitivity of the "off " contraction to cholinergic antagonists varies significantly among species that have been examined. In the opossum, "off " responses are minimally depressed ⁶ or enhanced ⁸ by atropine, whereasthey are completely abolished in the cat. ²³ The mediator(s) of residual (noncholinergic) "on" and "off " contractions have not been identified.

An understanding of the normal physiology of an organ should form the basis of a clinical approach to functional disorders of the organ. In the case of the esophagus, normal physiology has been poorly understood, limiting clinical perspective on primary esophageal disorders such as achalasia, diffuse spasm, and nutcracker esophagus. It is now appreciated that these apparently diverse motor disorders may represent distinct stages along a continuum of intramural neuropathy. ²⁴ Specifically, these diseases have in common a progressive breakdown of smooth muscle peristalsis and lower sphincter function possibly attributable to degeneration of inhibitory neuronal control mechanisms. This interpretation is supported by case reports of apparent transition from nutcracker esophagus to diffuse spasm ^{25, 26} and from diffuse spasm to achalasia. ^{24, 27}

By identifying a mediator of smooth muscle neuronal inhibition, the present study sheds light on the normal physiology of esophageal peristalsis. Specifically, the results suggest that the peristaltic wave is dependent on the coordination of cholinergic excitation with nitric oxide-dependent inhibition. The ability of the inhibitory component to mask the contractile effects of simultaneously present cholinergic excitation (as evidenced by the emergence of cholinergic "on" contractions during nitric oxide synthesis blockade) supports the view that a distally progressive release from inhibition could account for peristaltic propagation. This interpretation implies that neuronal excitation need not be aborally sequenced, but rather that a generalized cholinergic tone within the segment as a whole might modulate the amplitude of the contraction. With progressive deterioration of inhibitory innervation, high-amplitude peristaltic (nutcracker) and eventually simultaneous nonperistaltic (diffuse spasm) contractions would be expected as cholinergic excitation becomes less effectively balanced and controlled by the inhibitory mechanism. Manometric patterns consistent with diffuse esophageal spasm can be induced experimentally in some patients with nutcracker esophagus by bethanechol provocation. ²⁸ In achalasia, severe loss of postganglionic inhibitory innervation has been demonstrated, although cholinergic innervation is essentially intact. ^{29, 30}

Esophageal motility disorders are generally treated by mitigation of esophageal smooth muscle function either by calcium channel blockade or by myotomy. Identification of the inhibitory component as a major contributor to normal motility and of nitric oxide as a neurochemical mediator may allow for more tailored therapy. Future studies aimed at developing such therapy will involve assessment of nitric oxide synthase and contractile activity in diseased specimens.

Acknowledgments

We gratefully acknowledge the expert technical assistance of Kathleen A. Kee and the editorial assistance of Mary Sullivan Visciano. We also thank Dr. Michael Liptay for helpful comments on the manuscript.

Footnotes

From the Department of Surgery, Harvard Medical School, Brigham & Women's Hospital, Boston, Mass.,^a the Divisions of Respiratory Medicine and Cardiovascular Medicine, Duke University Medical Center, Durham N.C.,^b and the Department of Environmental Health, Harvard School of Public Health, Boston, Mass.^c

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): David J. Sugarbaker Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Richards, W. G. Articles by Sugarbaker, D. J. Search for Related Content PubMed PubMed Citation Articles by Richards, W. G. Articles by Sugarbaker, D. J.