Central nervous system nitric oxide induces oropharyngeal swallowing and esophageal peristalsis in the cat

GASTROENTEROLOGY 2000;119:377–385

Central Nervous System Nitric Oxide Induces Oropharyngeal Swallowing and Esophageal Peristalsis in the Cat MICHAEL J. BEYAK, SHUWEN XUE, PHILLIP I. COLLMAN, DIANA T. VALDEZ, and NICHOLAS E. DIAMANT Departments of Medicine and Physiology, Playfair Neuroscience Institute, Toronto Western Hospital, University of Toronto, Toronto, Ontario, Canada

Background & Aims: The functional role of brainstem nitric oxide (NO) in swallowing and esophageal peristalsis remains unknown. We examined the effects of blockade of central nervous system (CNS) NO synthase (NOS) on swallowing and on primary and secondary peristalsis. Methods: (1) The effect of intravenous (IV) NOS inhibitor NG-nitro-L-arginine (L-NNA) on swallowing and swallowing-induced peristalsis was examined. (2) An NOS inhibitor (NG-monomethyl-L-arginine [L-NMMA]) was administered into the fourth ventricle intracerebroventricularly (ICV), and its effects on swallowing and primary and secondary peristalsis were examined. Results: (1) IV L-NNA significantly reduced the number of oropharyngeal swallows and the induction of primary peristalsis in the smooth muscle portion of the esophageal body; the change was not significant within the striated muscle portion. (2) L-NMMA given ICV significantly reduced the number of oropharyngeal swallows and the incidence of primary peristalsis in both smooth and striated muscle, but the reduction in amplitude was significant only for the smooth muscle contraction. There was a significant reduction in both the amplitude and incidence of secondary peristalsis, only in the smooth muscle portion. Conclusions: CNS NO is an important neurotransmitter in the induction of oropharyngeal swallowing and esophageal peristalsis. The neural substrates mediating striated and smooth muscle peristalsis may be both anatomically and neurochemically distinct.

itric oxide (NO) is a novel neurotransmitter found throughout the central and peripheral nervous systems. Recent histochemical studies have provided evidence in cats,1,2 rats,3,4 rabbits,5 and humans6 that nitric oxide synthase (NOS) is localized in the nucleus of the solitary tract (NTS), nucleus ambiguus (NA), and dorsal motor nucleus of the vagus (DMV), regions implicated in the central control of swallowing and esophageal motility.7–9 Recently, NOS has been shown in the nucleus subcentralis of the NTS of rats and rabbits,4,5 the region believed to be responsible for the central programming of esophageal peristalsis, at least for the striated muscle

N

portion.7–9 Furthermore, a preliminary report showed that microinjection of an NO donor or L-arginine into the rostral DMV of the cat causes increases in lower esophageal sphincter (LES) pressure, the effect of L-arginine being inhibited by NG-nitro-L-arginine-methyl ester (L-NAME), an inhibitor of NOS.10 However, no functional studies have examined the role of NO in the central reflexes involved in the initiation of swallowing and regulation of primary and secondary esophageal peristalsis. We therefore examined the effects of NOS inhibition on oropharyngeal swallowing and esophageal peristalsis induced by swallowing and intraluminal balloon distention. We provide evidence that the L-arginine–NO pathway is implicated in the central control of swallowing and esophageal motility. A portion of this work has been published in abstract form.11,12

Materials and Methods The experimental protocol was approved by The Toronto Hospital Animal Care Committee. Short-term studies were performed on 31 cats of either sex weighing 2.8 – 6.0 kg. Ketamine hydrochloride (Rogarsetic, Rogar; 15 mg/kg intramuscularly [IM]) was used as an induction agent in all animals. In animals not undergoing superior laryngeal nerve (SLN) or craniotomy surgery, anesthesia was maintained using a continuous intravenous (IV) infusion of ketamine (15 mg 䡠 kg⫺1 䡠 h⫺1) using a volumetric infusion pump (IMED model 960; IMED Corp., San Diego, CA). After ketamine induction in the animals undergoing SLN surgery, urethane (ICN; 1.0 –1.3 Abbreviations used in this paper: CPG, central pattern generator; DMV, dorsal motor nucleus of the vagus; EAA, excitatory amino acid; EMG, electromyogram; ICV, intracerebroventricularly; L-NAME, NG-nitro-L-arginine methyl ester; L-NMA, methyl-L-arginine; L-NMMA, NGmonomethyl-L-arginine; L-NNA, NG-nitro-L-arginine; MH, mylohyoid; NA, nucleus ambiguus; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NMDA, N-methyl-D-aspartate; NOS, nitric oxide synthase; NTS, nucleus of the solitary tract; SLN, superior laryngeal nerve; UES, upper esophageal sphincter. © 2000 by the American Gastroenterological Association 0016-5085/00/$10.00 doi:10.1053/gast.2000.9308

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g/kg administered IV as 20% solution in water) was used for general anesthesia. Animals were intubated endotracheally to prevent airway obstruction and aspiration. Catheters were placed in the femoral arteries and veins of all animals for monitoring of blood pressure and administration of drugs, respectively. The internal branch of the SLN was isolated as follows. Through a ventral midline incision in the neck, the internal branch of the SLN was exposed and dissected free of the surrounding tissues, taking care not to disrupt its blood supply. The nerve was then placed over a set of silver wire electrodes (interelectrode distance, 2–3 mm), and the entire assembly was encased in vinyl polysiloxane (Reprosil; Dentsply Int., Millford, DE) to prevent current leak. Stimuli were delivered from a Grass S88 stimulator (Grass Instruments, Quincy, MA) through a Grass stimulus isolation unit (SIU 5) and constant current unit (CCU 1A). Stimulus parameters were as follows: pulse width, 500 milliseconds; intensity, 1–3 mA; frequency, 30 Hz; train length, 10 seconds. The effects of central administration of NOS inhibitors were examined in 12 cats. Dexamethasone, 0.1 mg/kg IM, was administered to each animal before surgery to prevent cerebral edema. The fourth ventricle was exposed as follows. The animal’s head was placed in a stereotaxic holder, a partial occipital craniotomy was performed, the meninges were divided with fine scissors, and the cerebellum was retracted, exposing the floor of the fourth ventricle. Drugs were administered into the fourth ventricle via a Hamilton Microliter syringe (Hamilton Company, Reno, NV), with the tip positioned just rostral to obex. NG-Monomethyl-L-arginine (LNMMA) was used as an inhibitor of NOS in these experiments because it is readily soluble at neutral pH and is free of antimuscarinic activity. The dosage of 5 mol/kg was chosen based on previous similar experiments that studied brainstem control of blood pressure in the rat. In all animals, the onset of the oropharyngeal stage of swallowing was monitored using bipolar electrodes implanted in the mylohyoid (MH) muscle. The electromyogram (EMG) signal was amplified using an HP 8811A bioelectric amplifier (Hewlett Packard Inc., Waltham, MA). Esophageal motility was monitored using a conventional perfused multilumen manometry catheter with side ports located 2, 4, 6, and 12 cm above the LES and a sleeve device to monitor LES events. A pressurized infusion pump (Mui Scientific, Mississauga, Ontario, Canada) was used to perfuse the catheter at a rate of 0.3 mL/min and the sleeve at 0.1 mL/min. The system was capable of recording a pressure increase of 300 mm Hg/s in the esophageal ports. MH EMG, arterial and esophageal pressures, and stimuli were simultaneously recorded on a chart recorder, 8-channel FM tape recorder (HP 3968A), and IBM AT-compatible computer using Extended Graphics Acquisition and Analysis version 3.5 (EGAA; RC Electronics, Santa Barbara, CA) dataacquisition software.

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Experimental Protocol At the beginning of each experiment, the esophagus was mapped manometrically, determining the length and location of the upper esophageal sphincter (UES), esophageal body, and LES. The smooth muscle portion of the esophagus was delineated with atropine methyl nitrate, 20 ␮g/kg, which eliminates smooth muscle peristalsis in the cat. Manometry ports 2 and 4 cm above the LES were always located in the smooth muscle portion, and the port 12 cm above the LES was located in the striated muscle portion of the esophagus. Swallowing and primary esophageal peristalsis were induced either with a 3-mL water bolus injected into the posterior pharynx or by stimulation of the afferent limb of the SLN. Secondary peristalsis was induced by inflation of a latex balloon (10 ⫻ 20 mm; maximum volume, 5 mL) connected to a 10-mL syringe with plastic tubing, positioned 5 cm above the LES. The balloon was inflated for 5 seconds with 5 mL of air. For each stimulus, a series of 7–10 control stimulations was made to induce swallowing, and oropharyngeal and esophageal responses were recorded. In the first group of 19 cats after the control period, 10⫺4 mol/kg NG-nitro-L-arginine (L-NNA) was administered IV, and the series of stimulations was then repeated. In 11 of these animals, 10⫺3 mol/kg L-arginine was administered IV and a series of 7–10 stimulations was repeated. In 9 animals in the second group, L-NMMA was administered intracerebroventricularly (ICV) after the control period, and the series of stimulation and/or balloon distentions was repeated. In 3 cats, the effects of D-NMMA, the inactive enantiomer, was also examined in a similar fashion. Velocity in the smooth muscle portion was calculated from the time delay between onset of the contraction wave at the recording ports 2 and 4 cm above the LES. Because only 1 recording site was present in the striated muscle portion, the time from the last EMG burst to the onset of the contraction at the striated muscle site was used as an indication of the propagation time in this portion.

Data Analysis The incidence of oropharyngeal swallowing and of striated and smooth muscle peristalsis under each experimental condition was calculated for each animal by dividing the total number of times a given response occurred by the total number of stimulus attempts; the incidence was expressed as a percentage. The number of oropharyngeal swallows (MH EMG bursts) evoked by each stimulus modality was counted and meaned for each experimental condition. Results are expressed as mean ⫾ SEM. The Student unpaired t test was used to compare means and test for statistical significance. The Fisher exact test was used to test for statistical significance between proportions. A P value of ⬍0.05 was considered statistically significant.

Drugs The following drugs were used in the present experiments: atropine methyl nitrate, L-arginine, L-NNA (Sigma, St. Louis, MO), L-NMMA, NG-monomethyl-D-arginine (Calbio-

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chem, La Jolla, CA), urethane (ICN), and ketamine hydrochloride (Rogarsetic; Rogar).

Results Swallowing Responses Evoked by Water Bolus or SLN Stimulation Swallowing was induced by injection of a 3-mL bolus of water into the posterior pharynx or stimulation of the SLN (30 Hz, 10 seconds). These stimuli evoked 1 of 3 different responses: (1) a complete swallow sequence including multiple MH EMG bursts, striated and smooth muscle esophageal peristalsis, and LES relaxation and contraction; (2) MH activity, smooth muscle peristalsis, and LES relaxation without striated muscle peristalsis; (3) MH activity and LES relaxation only, without esophageal body activity. Effect of IV L-NNA on Oropharyngeal Swallowing Responses L-NNA

was administered IV, and subsequent effects on swallowing responses induced by either SLN or water bolus stimulation were examined (Table 1). The swallowing response was marked by the appearance of the MH EMG bursts and quantified by the number of EMG bursts. In the control period, each stimulation of both water bolus and SLN induced the oropharyngeal swallowing response as evidenced by at least 1 EMG burst (therefore, the induction rate is 100% with both stimulation modalities). The induction rate was not significantly affected by IV L-NNA (95% and 100% after L-NNA, respectively). However, the number of EMG bursts evoked by either stimulus modality was reduced significantly by L-NNA administration (Figure 1). With water bolus–induced swallows, the MH EMG bursts were reduced from 8.9 ⫾ 0.8 to 2 ⫾ 0.2 (P ⬍ 0.05). Similarly, when swallows were evoked with an SLN stimulus, only 1.8 ⫾ 0.3 oropharyngeal swallows were Table 1. Effects of NOS Inhibition (L-NNA IV) on Induction of Oropharyngeal Swallowing and Peristaltic Contractions in the Striated and Smooth Muscle Esophagus Water bolus Control Swallow (%) Striated muscle (%) Smooth muscle (%)

L-NNA

IV

SLN

L-Arg

IV Control

L-NNA

L-Arg

IV

100

95

97

100

100

100

33

26

27

17

17

22

69

38a

41a

81

40a

38a

L-Arg, L-arginine. aP

IV

⬍ 0.05 compared with control, Fisher exact test.

Figure 1. Effects of IV L-NNA on the number of oropharyngeal swallows (MH EMG bursts) in response to water bolus– or SLN-induced peristalsis. The number of EMG bursts (swallows) was significantly reduced in both settings. The effects were not reversed by L-arginine administration. *P ⬍ 0.05 compared with control.

evoked, compared with control value of 10.5 ⫾ 0.6 (P ⬍ 0.05). Effect of IV L-NNA on Swallow-Induced Peristalsis The incidence of swallow-induced peristalsis was studied in both the striated and smooth muscle portions of the esophagus in the absence and presence of NOS inhibition by IV L-NNA (Table 1). L-NNA did not have a statistically significant effect on the incidence of striated muscle peristalsis induced by either water bolus (33% with control, 26% with L-NNA; P ⬎ 0.05) or SLN stimulation (22% control, 17% after L-NNA; P ⬎ 0.05; Table 1). In contrast, L-NNA did have a significant influence on the occurrence of peristaltic contractions in the smooth muscle portion of the esophagus. Whether water bolus stimulation or SLN stimulation induced primary peristalsis, L-NNA significantly reduced the incidence of primary peristalsis in the smooth muscle portion of the esophagus. In the presence of L-NNA, with a water bolus stimulus, esophageal peristalsis occurred only 38% of the time compared with a control value of 69% (P ⬍ 0.05). Similarly, with SLN stimulation, smooth muscle peristalsis occurred only 40% of the time; again, this value was significantly less than the control value of 81% (P ⬍ 0.05). L-NNA significantly reduced the amplitude of the contraction in the smooth (control, 30.7 ⫾ 3.3 mm Hg; L-NNA, 18.1 ⫾ 2.5 mm Hg; P ⬍ 0.02) but not in the striated (control, 20 ⫾ 2.2 mm Hg; L-NNA, 15 ⫾ 3.0 mm Hg; P ⬎ 0.05; Figure 2) muscle section. Velocity in the smooth muscle portion was increased (control, 3.1 ⫾ 0.5 cm/s; L-NNA, 6.7 ⫾

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Figure 2. Effects of IV L-NNA on amplitude of contraction with SLNinduced peristalsis. The amplitude in the smooth muscle section was significantly reduced, whereas the amplitude in the striated muscle section was not significantly affected. The reduced amplitude was not reversed by L-arginine administration. *P ⬍ 0.05 compared with control.

0.4 cm/s; P ⬍ 0.02; Figure 3). The time from the last MH EMG burst to the contraction in the striated muscle esophagus was not significantly altered by L-NNA (control, 3.6 seconds; L-NNA, 4.6 seconds; P ⬎ 0.05), indicating no major effect on velocity in this portion. Effect of IV L-Arginine on L-NNA–Mediated Effects In 11 cats, L-arginine (10⫺3 mol/kg) was administered after L-NNA. IV L-arginine was unable to reverse the effects of L-NNA on SLN- or water bolus–induced swallowing and esophageal peristalsis (Table 1 and Figures 1 and 2), except for the velocity of peristalsis, which became similar to the control value (3.8 ⫾ 0.6 cm/s; Figure 3). Effect of ICV L-NMMA on Oropharyngeal Swallowing To examine the effect of inhibition of central NOS on swallowing, L-NMMA (5 ⫻ 10⫺6 mol/kg) was administered ICV. For these experiments, swallowing was induced by SLN stimulation only. Similar to the effects of IV L-NNA, ICV L-NMMA did not significantly affect the induction of oropharyngeal swallowing (Table 2). However, the number of MH EMG bursts evoked per SLN stimulus was significantly reduced from 1.78 ⫾ 0.14 to 1.26 ⫾ 0.16 (P ⬍ 0.05). Effect of ICV L-NMMA on SLN-Evoked Esophageal Peristalsis ICV L-NMMA resulted in significant inhibition of the induction of primary peristalsis. The incidence of

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Figure 3. Effects of IV L-NNA and ICV L-NMMA on the velocity of SLN-induced peristaltic contractions. The peripherally administered NOS inhibitor (L-NNA) significantly increased the velocity of peristaltic contractions in the smooth muscle section; this effect was readily reversed by IV L-arginine. However, the centrally administered NOS inhibitor (L-NMMA) had no effect on the velocity of peristaltic contractions (pooled data from both SLN- and balloon distention–induced peristaltic contractions in smooth muscle section). *P ⬍ 0.05 compared with control.

peristalsis in striated and smooth muscle sections was 22% and 43% in the control period, respectively. ICV L-NMMA significantly inhibited the incidence of peristalsis in both striated and smooth muscle sections (14% and 20% after ICV L-NMMA, respectively; P ⬍ 0.05; Table 2). This effect was seen within 10 –15 minutes after L-NMMA administration. In these same experiments, the effect of ICV L-NMMA on the amplitude of the evoked peristaltic wave was also examined. The amplitude of contraction in the striated muscle was not significantly affected (control, 19.2 ⫾ 3.8; L-NMMA, 21.6 ⫾ 6.5 mm Hg). However, ICV L-NMMA significantly reduced the amplitude of contraction in the smooth muscle from 22.4 ⫾ 4.8 mm Hg with control to 9.8 ⫾ 3.2 mm Hg after L-NMMA (P ⬍ 0.05).

Table 2. Effects of NOS Inhibitors (L-NMMA ICV) on Induction of Oropharyngeal Swallowing and Peristaltic Contractions in the Striated and Smooth Muscle Esophagus SLN Control Swallow (%) Striated muscle (%) Smooth muscle (%) aP

100 22 43

Balloon distention

L-NMMA

100 14a 20a

ICV

Control — 22 75

⬍ 0.05 compared with control, Fisher exact test.

L-NMMA

— 14 54a

ICV

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Table 3.

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D-NMMA ICV Has No Effect on Oropharyngeal Swallowing and Esophageal Peristalsis Induced by SLN or Balloon Distention

SLN Control Swallows (%) MH EMG bursts Incidence of peristalsis (%) Striated muscle Smooth muscle Contraction amplitude (mm Hg) Striated muscle Smooth muscle

Balloon distention D-NMMA

ICV

Control

D-NMMA

ICV

100 1.78 ⫾ 0.1

100 1.75 ⫾ 0.4

— —

— —

22 38

24 36

20 68

23 64

21 ⫾ 3.3 25 ⫾ 2.1

19 ⫾ 1.7 20 ⫾ 2.4

22 ⫾ 3.4 29 ⫾ 3.1

24 ⫾ 1.6 30 ⫾ 4.3

Effect of ICV L-NMMA on Balloon Distention–Evoked Esophageal Peristalsis In the cat, secondary peristalsis is dependent on intact central nervous system (CNS) vagal connections. Therefore, we examined the effect of ICV L-NMMA on the secondary peristalsis induced by balloon distention (Table 2). Evoked peristalsis in the striated muscle esophagus was infrequent and not significantly changed after the administration of ICV L-NMMA (22% vs. 14%; P ⬎ 0.05). There was no statistically significant effect on the amplitude of contraction in the striated muscle portion (25.1 ⫾ 3.3 vs. 16.6 ⫾ 2.8 mm Hg; P ⬎ 0.05). In contrast, the incidence of secondary peristalsis in the smooth muscle esophagus was significantly reduced from 75% to 54% (P ⬍ 0.05) after ICV L-NMMA. In 2 cats it was completely abolished. This effect occurred within 10 –15 minutes after administration of L-NMMA. The amplitude of contraction was also reduced from 27.4 ⫾ 3.7 to 10.9 ⫾ 3.3 mm Hg (P ⬍ 0.02). There was no significant effect on peristaltic velocity with either balloon- or SLN-induced esophageal peristalsis in the smooth muscle section (3.8 ⫾ 0.2 vs. 3.6 ⫾ 0.3 cm/s; Figure 3). Effect of ICV D-NMMA on Swallowing and Esophageal Peristalsis To demonstrate the stereospecificity of the effects of monomethyl arginine on swallowing and esophageal peristalsis, the inactive D-enantiomer (D-NMMA) was administered to 3 cats and similar experiments were repeated. There was no significant effect of D-NMMA on oropharyngeal swallowing response, incidence of esophageal peristalsis, or contraction amplitude, regardless of the stimulus used. The results are summarized in Table 3. The peristalsis velocity in the smooth muscle was also not affected (control, 2.8 ⫾ 0.16 cm/s; D-NMMA, 2.7 ⫾ 0.24 cm/s).

Discussion This study shows that NO is a functional neurotransmitter in the central pattern generator (CPG) responsible for swallowing and the central control of esophageal peristalsis. The number of swallows induced by afferent stimulation (a water bolus or SLN stimulation) is readily reduced by central NOS inhibition. The initiation of primary or secondary peristalsis in the smooth muscle esophagus is also significantly reduced, as is the smooth muscle contraction amplitude. Under these experimental conditions, striated muscle peristalsis is less or little affected by NOS inhibition. The study also shows that peripherally administered L-NNA can have effects on centrally mediated swallowing and primary and secondary esophageal peristalsis. Peripherally administered NOS inhibitors, including L-NNA,16 –18 methyl-L-arginine (L-NMA), and L-NAME,19 can access structures within the blood– brain barrier to affect neuronal activity and physiologic function. The maximal central effect of IV L-NAME on central neuronal activity in the NTS of the rat takes 12–15 minutes to occur and the same, more prolonged time is required to reverse this effect with IV L-arginine.19 Our previous studies in the cat showed that IV L-NNA not only increased peristaltic velocity in the distal smooth muscle esophagus within 5 minutes but also decreased distal contraction amplitude, the latter occurring much more slowly (approximately 20 minutes).20 The velocity change was quickly and completely reversed by L-arginine, compatible with a peripheral site of action of the L-NNA. However, the decrease in amplitude was slowly and not readily reversed, suggesting that L-NNA was in significant part acting to reduce amplitude at other than a peripheral site, perhaps centrally. Similar effects of IV L-NNA and L-arginine were seen in the present experiments. Furthermore, that IV L-NNA can mimic the central effects of ICV L-NMMA further supports the

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notion that peripherally administered NOS blockers have significant action at central sites, in this case on those neural mechanisms that use NO as a neurotransmitter and are involved with control of swallowing and esophageal peristalsis. As in previous studies, IV L-NNA increased peristaltic velocity in the smooth muscle esophagus. The lack of effect of ICV L-NMMA on velocity indicates that any central control of velocity by NO in either smooth or striated muscle sections is minimal, and the effect of IV L-NNA on velocity is primarily a peripheral action. The lack of central effect of L-arginine in our experiments may indicate that in certain instances L-NNA is more tightly or irreversibly bound to the central NOS. This study represents the first demonstration of a functional role for NO in the initiation of oropharyngeal swallowing. Both L-NMMA applied to the floor of the fourth ventricle and IV L-NNA significantly reduced the average number of swallows elicited by a water bolus or SLN stimulation by up to 80%. The brainstem location for optimum initiation of the swallow has been localized to the dorsomedial medulla with bilateral representation and anteroposterior position between the facial nucleus and the middle of the inferior olive.7,21–24 The detailed circuitry of the CPG components involved in this initiation process is not yet known. The afferent inputs that normally initiate the swallow, largely carried in the SLN, converge onto the solitary tract in the caudal medulla. In the cat, most sites that are responsive to direct stimulation are clustered in the caudal brainstem at the level of the obex and in the dorsal reticular formation ventral and lateral to the solitary tract. As noted above, previous studies have documented the presence of NO in the brainstem regions implicated in the control of swallowing in a number of species, including the cat.1– 6 Those afferents arise from the striated muscle esophagus synapse on premotor neurons in the central subnucleus of the NTS, which innervate esophageal motor neurons in the compact formation of the nucleus ambiguous. These premotor neurons contain NOS, and presumably similar NTS neurons in other subnuclei receiving input from the pharynx25,26 and cerebral cortex to initiate swallowing also contain NOS. NOS inhibition in our experiments alone did not completely block oropharyngeal swallowing activity, suggesting that other neurotransmitters play an important role in initiating this vital function. These probably include excitatory amino acids (EAA) activating both N-methyl-D-aspartate (NMDA) and nonNMDA receptors27–29 and perhaps other neurotransmitters such as catecholamines, serotonin, thyrotropin-releasing hormone, vasopressin, and oxytocin.

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Present concepts picture the CPG for swallowing and esophageal peristalsis as a serial network of linked neurons within the NTS and neighboring reticular formation, such that once it is activated, its rostrocaudal organization produces sequential excitation of motor neurons serving muscles along the deglutition pathway.7,23 At least 2 subnetworks are considered to be present, one for the oropharyngeal phase and the other for the esophageal phase of swallowing.29,30 Even segmental subcircuits have been suggested for the striated muscle portion.31 The present studies show for the first time that initiation of primary peristalsis in the esophagus is sensitive to NOS inhibition, the action most prominent in the smooth muscle portion. IV L-NNA and ICV L-NMMA were able to reduce the incidence of smooth muscle peristalsis induced by swallowing and/or SLN stimulation by more than 50%, whereas the reduction in the striated muscle portion was small and not usually significant. However, the induction of peristalsis in the striated muscle portion was also consistently and significantly less than that in the smooth muscle portion, regardless of the stimulus modality used for initiation of swallowing. These observations suggest that the central mechanisms that mediate peristalsis in the striated and smooth muscle portions of the esophagus differ not only neuroanatomically, but also neurochemically. For the striated muscle esophagus of the rat and rabbit, premotor neurons of that part of the subnucleus centralis of the NTS that receives esophageal afferent input have NOS and send reduced nicotinamide adenine dinucleotide phosphate (NADPH)–reactive projections to the compact subnucleus of the NA, where the esophageal motoneurons are located.4,5 These esophageal premotor neurons also receive input from pharyngeal premotor neurons in the intermediate and interstitial subnuclei of the NTS and connect with third-order esophageal neurons in multiple nuclei of the reticular formation, including the parvocellular nucleus.30 The reticular formation nuclei contain interneurons that are active as part of the CPG control for swallowing and esophageal peristalsis.9,32 In some species, sensory reinforcement from the esophagus is important for both initiation and shaping of swallow-induced contraction of the striated muscle esophagus.31,33 That is, the subnucleus centralis neurons that receive esophageal afferent input probably serve a number of interrelated functions: (1) as a portal for initiation of the programmed peristaltic sequence within the CPG circuitry, whether centrally or peripherally initiated; (2) as a communication pathway within the serial network of linked neurons within the NTS; and (3) as a sensory-motor pathway for modulation

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of the programmed activity once initiated. The role played by NO in any of these functions is unknown. For example, the time delay to onset of the swallow indicates that afferent information initiating the swallow is fed into the CPG from the centralis neurons before direction is given to the motor neurons to produce a contraction. There is no information on whether this direction to the motor neurons returns via the NO-laden subnucleus centralis neurons or via a different pathway that may or may not use NO.34 In our experiments, IV L-NNA or ICV L-NMMA had a small, usually insignificant effect on the initiation of peristalsis in the striated muscle esophagus and no significant effect on the amplitude of the contractions. Therefore, surprisingly, we did not demonstrate a definite or consistent role for NO in initiation of peristalsis or in regulation of contraction amplitude in this portion. It is not clear if this finding reflects a limited role for NO in the face of other neurotransmitters known to be important, is attributable to species differences, or is a result of the experimental condition. Many different neurotransmitters are involved in these functions.8,35,36 For example, muscarinic cholinergic excitation facilitates the initiation of peristalsis in the striated muscle portion of the rat, whether centrally29,37 or peripherally38 induced, although excitatory amino acids appear to be most important.38 On the other hand, activation of ␥-amino butyric acid receptors inhibits the oropharyngeal-esophageal linkage and initiation of esophageal peristalsis in the rat.39 There is no specific information on the neurotransmitter content, including that of NOS, in the premotor and putative interneurons in the cat, which could conceivably differ from that seen in the rat. Finally, the low incidence of peristalsis in this portion, as opposed to that in the smooth muscle, may indicate a more selective effect of conditions, such as anesthesia, that impair one or more of the interacting neurotransmitters necessary for striated muscle peristalsis and act to augment the action of one another. Such a synergistic effect of the interaction between NO and vasoactive intestinal polypeptide has been postulated for their action to relax the LES.40 Clarification of the role of NO is open to further study. Information on various putative neurotransmitters involved in the programming of peristalsis is not available for the smooth muscle portion, which receives its motor input from neurons in the DMV. Our studies suggest that initiation and programming of peristalsis in the smooth muscle portion involves NO in some way, and this involvement is more prominent than that of the striated muscle portion.

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Motor neurons to the smooth muscle esophagus, including the LES, are located in 2 subregions of the DMV.41 Evidence from studies of the cat LES suggest that the rostral region contains neurons that mediate excitation, and the caudal region contains neurons that mediate inhibition of the esophagus.42 Furthermore, microinjection of an NO donor or L-arginine into the rostral group causes an increase in LES pressure, the effect of L-arginine being prevented by NOS inhibition.10 In addition, in vitro recordings from rat DMV neurons have shown that these neurons can be excited by NO, NO donors, and L-arginine and that NOS inhibition both decreases the rate of spontaneous discharge of these neurons and prevents the effect of L-arginine.43 DMV motor neurons in the cat are also activated by the EAA glutamate; as elsewhere in the nervous system, the effects of glutamate can be mediated through the release of NO.44 Our finding that NOS inhibition significantly reduced amplitude of the contraction in the smooth muscle but not the striated muscle portion of the esophagus suggests that activation of the motor neurons to the 2 regions may also be different. Those in the DMV and serving the smooth muscle may rely more on EAA release of NO, whereas the NA motor neurons serving the striated muscle may rely more on something other than the well-documented nitrergic innervation from the subnucleus centralis. Previous studies in the cat showed that secondary peristalsis is abolished by vagal cooling and therefore is dependent on intact connections between the esophagus and CNS.15 We provide evidence that pharmacologic manipulations of the NO pathway within the CNS can also inhibit the induction of secondary esophageal peristalsis, supporting the idea that secondary peristalsis in the cat is centrally mediated. Furthermore, our studies indicate that as for the induction of primary peristalsis, NO is probably an important neurotransmitter in this process. The balloon was distended in the distal esophagus, primarily in the smooth muscle portion, and ICV L-NMMA reduced the induction of motor activity in both striated and smooth muscle portions. It is not clear whether the effect on the striated muscle portion reflected the induction of the reflex activity through those neural pathways serving predominantly the smooth muscle portion and therefore more sensitive to NOS inhibition. As with the induction of primary peristalsis, secondary peristalsis was not completely abolished, indicating that other neurotransmitters such as excitatory amino acids are important.38 In summary, the present studies indicate that NO is a functional neurotransmitter in the central control mech-

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anism for initiation of swallowing and control of primary and secondary esophageal peristalsis. The role of NO in control of peristalsis seems to be most prominent for the smooth muscle portion and includes regulation of contraction amplitude in this region. The findings also suggest that the central mechanism for initiation and regulation of peristalsis in the striated and smooth muscle portions of the esophagus may differ not only neuroanatomically, but also neurochemically. The presence and functional importance of central NO may have disease implications that require further study. For example, achalasia of the esophagus is associated with a deficiency or absence of peripheral neural NOS,45 and a similar central deficiency of NOS in damaged brainstem neurons46 could contribute to the clinical picture. Finally, the interpretation of the findings of previous and future studies in which NOS blockers are administered peripherally and alter esophageal contraction and peristalsis must include consideration of the central effects of these agents.

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Received June 3, 1999. Accepted March 15, 2000. Address requests for reprints to: Nicholas E. Diamant, M.D., Toronto Western Hospital, McLaughlin Pavilion, Room 12-419, 399 Bathurst Street, Toronto, Ontario M5T 2S8 Canada. e-mail: [email protected]; fax: (416) 603-6204.