Abstract
AIM To observe the effect of exogenous nitric oxide (NO) on antralcircular muscle motility of rat stomach and to analyze preliminarily its mechanism.
METHODS Rats wereanesthetized with urethane. The antral circular muscle strips were prepared by theconventional method. The motility of muscle strips was recorded by an isotonic transduceron a pen-recorder. Exogenous NO was prepared by chemical reaction between nitric acid andcopper.
RESULTS Exogenous NOsignificantly inhibited spontaneous and acetylcholine-induced motility of the musclestrips (P<0.01), but acetylcholine did noteliminate NO-induced inhibition (P>0.05).The NO-induced inhibition was not affected by atropine, propranolol, phentolamine or TTX.However, the inhibition was eliminated by oxyhemoglobin.
CONCLUSION Exogenous NOacting on the antral circular smooth muscle of rat stomach has the same effect as nonadrenergic, noncholinergic (NANC) inhibitory nerve on the muscle motility. Theinhibitory effect of exogenous NO is induced through its direct action on the smoothmuscle cells.INTRODUCTION
Furchgott and Zawadzki^[1] were the first to find that endothelium-derived relaxingfactor (EDRF) mediated acetylcholine-induced relaxation of vascular smooth muscles. Sincethen a number of studies have indicated that EDRF is nitric oxide (NO)^[2,3] . Recentstudies have provided evidence that NO may also participate in regulating several otherphysiological processes^[4,5] . Bred and co-workers^[6] reportedthat NO synthase is localized in the myenteric plexus and in the neuronal processes withinthe circular muscle layer of the rat intestine. Since then much compelling evidence hasindicated that NO acts as a NANC neurotransmitter in the gastrointestinal tract^[7-9] . However,this viewpoint is not yet supported by experiments on gastric antrum. In addition, littlework has been done to study the effects of exogenous NO on the motility of gastric fundus^[9] . On theother hand, in studying the physiological role of NO in gastrointestinal tract, sodium nitroprus-side and other nitrogen oxide-containing drugs were usually used as a nitricoxide donator. However these studies neglected the effect of metabolites of these drugs onthe gastrointestinal motility. The aim of the present study was to observe whetherexogenous NO imitates the effect of NANC inhibitory nerve on the antral circular musclemotility of rat stomach, and to figure out preliminarily its mechanism.

MATERIALS AND METHODS

Wistar rats of both sexes wereanesthetized with urethane (1g/kg). The abdomen of each animal was opened along themidline. Stomach was removed and placed in a pre-oxygenated Tyrode solution. Then themucosal layer was removed. The long axis of the stomach was cut parallel to the circularmuscle fibers and the strips of circular muscle layer (2mm× 10mm) wereprepared. The muscle strip was placed in a chamber (10ml volume) containing Tyrodesolution. The solution was constantly provided with oxygen and maintained at 37℃± 0.5℃. One end of the strip was fixed to the floor of thechamber and the other end was attached to an isotonic transducer (TD112S, NIHON KOHDEN) torecord motility of the muscle strip. The motility of the strips was recorded successivelyon the polygraph (RM6200, NIHON KOHDEN).
No gas was prepared by chemical reaction between 30%-35% nitric acid and copper usingKipps apparatus and purified through sodium hydroxide solution. The apparatus was cleansedof O₂ by bubbling it with N₂ through air-tight tube for 60min. NOgas was bubbled (about 100-120 bubbles/min) into 8ml of Tyrode solution for 20min to yieldapproximately 1mM solution. In our experiment, the relative concentration of NO wasexpressed by bubbling time. For stimulation of the muscle strips, we directly bubbled NOinto the chamber solution through a needle fixed to a position approximately 5mm from thestrip.
Oxyhemoglobin was prepared as hemolysate by a modified method of Bowman et al^[10] . Wholeblood was collected from tail veins of the rat and placed into glass tubes containingsodium heparin. The red blood cells were separated by centrifugation, the plasma and buffcoat were removed, and the red blood cells were washed three times with Tyrode solution.Washed red blood cells (1ml) were then added to 4ml of hypotonic solution to lyse thecells. Cell membranes were removed by centrifugation at 6000× g for 120min.The concentration of hemoglobin in the supernatant was measured spectrophotometrically as methemoglobin. The hemoglobin solution was either used immediately or stored frozen at -20℃for use within 24h. Solutions containing primarilymethemoglobin were prepared by dissolving purchased hemoglobin.
The Tyrode solution used in this study contained (mmol/L): NaCl 147, KCl 4, CaCl₂ 2, MgCl₂ 1.05, NaH₂ PO₄ 0.42, Na₂ HPO₄ 1.81, glucose 5.5 pH was regulated to 7.4 and the solution was bubbled with oxygen. Drugsused were atropine sulfate, hemoglobin from human erythrocytes, propranolol hydrochloride,phentolamine hydrochloride and tetrodotoxin (TTX) (all from Sigma Chemical Co., USA).Concentrations given in the results were the final concentrations of the substances in thechamber. N₂ was purchased from a freezing material plant, Yanji, China.
Quantitative data were presented as. Individual strip was exposed toexperimental intervention only once. The frequency and amplitude of the motility beforethe experiment were compared with those after experimental treatment, and mean changeswere evaluated by paired t test. A possibility of 0.05 or less was consideredstatistically significant.

RESULTS
Effects of NO on spontaneous motility

After incubation ofthe muscle strips for 30min approximately, spontaneous motility of the strips wasobserved. The phasic contractions were 4.5c/min±0.2c/min in frequency and 0.44g±0.06g inamplitude (Table 1). Administration of exogenous nitric oxide inhibited both tonic andphasic contractions in time-dependent manner. There was no exception in all 30 cases. Itcan be seen from Figure 1 that NO-induced basic tone descended temporarily and slightly,while the phasic contractions were eliminated completely. The latency of exogenous nitricoxide action was less than 6s. N₂ gas did not inhibit the motility (n=12,P>0.05).

The effect of NO onacetylcholine-induced excitatory motility

Treatment withacetylcholine (10μmol/L) increased themotility of the strips. However, nitric oxide inhibited the acetylcholine-inducedexcitatory motility. Both basic tone and phasic contraction were inhibited. In 8 cases,there was no exception. Figure 2 shows that basic tone was significantly and temporarilydecreased, indicating that inhibitory action of NO became stronger when motility wasincreased. On the contrary, NO-induced inhibition was not affected by acetylcholine(Figure 2).

Effects of drugs on theNO-induced inhibition

In order to study thecharacteristics of the NO-induced inhibition, we observed the effects of drugs on theNO-induced inhibition. NO-induced inhibition of antral circular muscle motility was notaffected by atropine (1μmol/L) (Figure 3).
It can be seen from Figure 3 thatNO-induced inhibition of antral circular motility was not affected by adrenergic receptorblockers of phentolamine and propranolol. It was also not affected by tetrodotoxin either.In addition, hemoglobin (7.4μmol/L) usuallyinduced elevation of the basic tone of the muscle strips, while the amplitude of phasiccontraction was decreased. The effect of hemoglobin on the motility was blocked by nitricoxide. However, NO-induced inhibition was eliminated by the presence of oxyhemoglobin.

Table 1 The effects of drugs on the NO-induced inhibitory motilityof antral circular muscle

Groups	n	Frequency (c/min)		Amplitude (g)
		Before	After	Before	After
Spontaneous	30	4.5±0.2		0.44±0.06
NO	30	4.5±0.2	0b	0.44±0.06	0b
Ach+NO	8	8.0±0.5	0b	1.00±0.29	0b
NO+Ach	8	0	0	0	0
Atro+NO	8	4.3±0.3	0b	0.28±0.05	0b
Phent+NO	4	3.9±0.4	0b	0.43±0.11	0b
Pro+NO	4	4.7±0.2	0b	0.25±0.08	0a
TTX+NO	4	3.7±0.5	0b	0.65±0.13	0a
Hb+NO	9	3.7±0.5	3.2±0.4	0.41±0.07	0.39±0.06

Note: compared with data before treatment:a P<0.05；b P<0.01.

Figure 1 Nitric oxide inhibits spontaneous motility of the antral circular muscle in a time-dependent manner.
Figure 2 The effect of NO on acetylcholine-induced excitatory motility (left) and the effect of acetylcholine on NO-induced inhibitory motility of the strips (right).
Figure 3 Effects of drugs on inhibitory action of NO.

It is known that inhibitory innervation to the sto-mach plays animportant role in the receptive rela-xation of the stomach. A number of studies showedthat part of the inhibitory nerves distributed in the stomach were non-adrenergic andnon-cholinergic (NANC) inhibitory nerves, but the identification of the NANC inhibitoryneurotransmitter remained unclear^[11] . Of the putative NANC inhibitory neurotransmitters studied, ATPand VIP are the substances for which more studies have been done, but the evidence isincomplete. Recently, a non-purinergic and non-peptidergic system utilizing NO as thefinal transmitter has been found to mediate NANC neural inhibition in the rat gastricfundus^[12] .
The results in this study showed that exogenous NO strongly inhibited spontaneous phasiccontraction and lowered the basic tone of the muscle strips slightly and transiently.After exposure of the strips to oxyhemoglobin, we observed that the basic tone of themuscle strips increased while the amplitude of the spontaneous phasic contractiondecreased. However, the effect of oxyhemoglobin was eliminated by exogenous NO. Theresults indicate that there is a tonic release of endogenous NO in the gastric antrum. Ourresults provide evidence for a physiological role of NO in gastric antral motility. Ozakiand co-workers^[13] indicated that inhibitory modulation of phasic contractionsresulted from continuous spontaneous release of NO in the canine gastric antrum. A recentstudy also demonstrated that vagus-mediated relaxation of the rat stomach was mediated byNO^[14] . In brief, it appears that inhibition of gastric smooth musclemediated by NO plays an important physiological role. It also indicates that there is aconstant release of NO in the gastric antrum, at least in the rat stomach.
Th results also showed that NO-induced inhibition of the motility was not affected byatropine, phentolamine or propranolol, and exogenous NO imitated the effect of stimulationof NANC inhibitory nerves on the gastric antral motility. However, Stark and co-workers^[15] reported thathyperpola-rization of the circular muscle of canine jejunum induced by NANC inhibitorynerve was different from that induced by exogenous NO.
If we presume that exogenous NO mediates the NANC nerve inhibition, a question arises:where is the likely site of NO action One possibility is that NO acts at the interneuronsite, releasing another NANC inhibitory transmitter to act on target smooth muscle cells.Another possibility is that NO acts directly on target smooth muscle cells. Our resultssupported the latter possibility because the response to exogenous nitric oxide was notaffected by the presence of tetrodotoxin. To be exact, the site of action of exogenous NOcould be in the cell. In other words, NO acts on the part posterior to the M receptor inexcitation-contraction coupling mechanisms of acetylcholine, because acetylcholine-inducedexcitation was eliminated by exogenous NO, but acetylcholine did not eliminate NO-inducedinhibition.
In summary, the present study shows thatexo-genous NO acting on the antral circular smooth muscle of the rat stomach imitated theeffects of NANC inhibitory nerve on the gastric antral motility. The inhibitory effect maybe induced through its direct action on the smooth muscle cells. Thus, the possibilitythat NO acts as a modulator can not be excluded.

Research Laboratory of Gastrointestinal Physiology,Yanbian University College of Medicine, Yanji 133000, Jilin Province, China
Tel. +86· 433· 2660586
JIN Nan-Ge, male, born on 1970-04-09 in Yanji, Chinese-Korean Nationality, entered YanbianMedical College as a candidate for master degree in 1995, having 2 papers published.
Project supported by the National Natural Science Foundation of China,No.39660026.
Correspondence to: Dr. LI Zai-Liu, Research Laboratory ofGastrointestinal Physiology, Yanbian University College of Medicine, 121 Juzi Jie, Yanji133000, Jilin Province, China
Received 1997-07-10

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