Departments of 1 Surgery and
2 Gastroenterology, Inhibitory nitrergic neurons are known to play a
role in the regulation of motility patterns of the distal esophagus,
the lower esophageal sphincter (LES), and the gallbladder. Our study aim was to investigate the effects of "long-term" (i.e.,
prolonged) oral intake of
L-arginine
(L-Arg), the endogenous source
for nitric oxide (NO) synthesis, on postprandial LES pressure (LESP),
esophageal motility, gastroesophageal reflux, and gallbladder
motility. L-Arg (30 g/day) or glycine (placebo; 13 g/day; isosmolar) was given orally to 10 healthy male volunteers for 8 days, according to a randomized,
crossover design. Twenty-four-hour urinary nitrite/nitrate excretion
was measured to indicate NO synthesis. Basal early postprandial LESP
was lower after L-Arg ingestion
(2.2 kPa) than after glycine ingestion (2.7 kPa)
(P < 0.05).
L-Arg abolished the
physiological late postprandial rise in LESP. Transient LES relaxations
were longer lasting after L-Arg
ingestion (P < 0.02). Esophageal
motility and reflux were not affected (not significant). Fasting and
residual gallbladder volumes were greater after
L-Arg ingestion
(P < 0.05). Urinary nitrite/nitrate
excretion was higher after L-Arg
intake (P < 0.05). In conclusion,
long-term oral L-Arg suppresses
late postprandial LESP increase, prolongs transient LES relaxations, and increases fasting and residual gallbladder volumes. These effects
may be mediated by increased NO synthesis.
nitric oxide; lower esophageal sphincter; nitrate
NITRIC OXIDE (NO), produced from
L-arginine by enzymatic
oxidation of a terminal guanidino nitrogen atom of
L-arginine by the enzyme NO
synthase (NOS), is involved in a number of biological actions. NO was
originally shown to act as a relaxing agent in vascular tissue, with NO
formed by an enzyme in vascular endothelial cells (28). NO is now known
to be formed in several other cells and tissues, e.g., macrophages and
neurons, involving different isoforms of NOS (24). NO plays an
important role in the regulation of gastrointestinal motility, serving
as a neurotransmitter in nonadrenergic noncholinergic (NANC) pathways
of the gastrointestinal tract (32).
Excitatory cholinergic and inhibitory NANC nerves are known to play a
role in the peristalsis of the esophageal body (1) and in the
relaxation of the lower esophageal sphincter (LES) (4). There appears
to be a gradient of decreasing cholinergic and increasing NANC
influence along the esophagus in the aboral direction (1). NO is a
mediator of LES relaxation induced by swallowing, esophageal
distension, and vagal efferent nerve stimulation (29, 40). In the
esophageal body, NO is involved in the latency period and latency
gradient as well as in the contraction amplitude of esophageal
peristalsis, especially in the distal part of the esophagus (1, 14,
41). The L-arginine-NO pathway
may also play a role in transient LES relaxations (TLESRs), i.e.,
abrupt decreases in LES pressure (LESP) to the level of intragastric pressure that are not triggered by swallowing (23) and that are the
main mechanism underlying gastroesophageal reflux (7, 33).
The L-arginine-NO pathway is
involved in the regulation of gallbladder motility (9, 25); gallbladder
muscle relaxation is also mediated by NANC inhibitory nerve activity
(21).
In vivo studies on the esophagus and gallbladder have so far only
concerned the effects of intravenous
L-arginine or NO donors or the
effects of NOS inhibitors with subsequent reversion of the inhibitory
effect by intravenous
L-arginine. The effects of prolonged oral administration of
L-arginine on both the esophagus and the gallbladder have not been studied.
Therefore, our aims were 1) to
investigate whether "long-term" (prolonged administration, as
opposed to single bolus or infusion) oral
L-arginine intake increases NO
production in healthy humans, 2) to
investigate whether these increased NO levels affect esophageal motility, LESP, TLESRs, and the occurrence of gastroesophageal reflux,
and 3) to study the effects of
increased NO levels on gallbladder motility.
First we performed a pilot study with a randomized, double-blind,
placebo-controlled, crossover design aimed at verifying that glycine
has no effects on the esophagus and gallbladder and could therefore be
used as an amino acid placebo in a subsequent L-arginine study.
The effects of the amino acid glycine on esophageal motility, LESP,
basal gallbladder volume, and gallbladder emptying were evaluated. Six
healthy male volunteers [age 27.0 ± 3.2 years (mean ± SD); body mass index 23.0 ± 2.7 kg/m2] participated. Glycine
and a placebo solution, identical to the glycine solution as to caloric
content (using glucose as a caloric substrate for the placebo
solution), chloride amount, osmolarity, pH, taste, and volume, were
given as a drink (300 ml/day) four times a day for 7 days, with a
washout period of at least 7 days. Esophageal motility and LESP were
measured after 6 days of glycine or placebo intake, after a
standardized meal (2,810 kJ; 30 g fat, 30 g protein, and 70 g
carbohydrates), by using stationary esophageal manometry.
Fasting gallbladder volume was measured using ultrasonography both
before and after glycine or placebo intake. Residual gallbladder volume
was measured only after 6 days of glycine or placebo intake. No
significant differences were observed in esophageal motility, LESP, and
TLESRs between glycine and placebo ingestion. Gallbladder volumes
(fasting, residual, and ejection) were not significantly different
between glycine and placebo ingestion either
(P > 0.05). We thus concluded that
glycine in an oral dose of 13 g/day for 7 days does not significantly
affect either esophageal or gallbladder motility. The lack of a glycine
effect on the esophagus and LES is consistent with the findings of
McCallum et al. (20). Therefore, glycine was chosen as a suitable
placebo for the study of the effects of
L-arginine.
Protocol.
Ten other healthy male volunteers [age 24.2 ± 4.1 years (mean ± SD); body mass index 22.1 ± 3.3 kg/m2], who were nonsmokers
and not on medication, received the amino acids
L-arginine and glycine, each
over an 8-day period, according to a randomized double-blind crossover
design. A washout period of 6 days was allowed between the 8 days of
amino acid ingestion. Figure 1 shows a
scheme of the protocol.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
View larger version (23K):
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Fig. 1.
Scheme of the study protocol with a randomized, crossover design. Solid
bars indicate periods of amino acid intake
[L-arginine
(L-Arg) or glycine (Gly)].
All measurements are shown with their corresponding days in the
protocol. GBV, measurement of gallbladder volume.
Amino acid drinks. Solutions of the amino acids L-arginine and glycine were prepared by the pharmacy at the University Hospital Utrecht. The composition of the daily dose was as follows: 36.28 g L-arginine-HCl (30 g L-arginine) or an isosmolar amount of glycine-HCl (19.31 g; 13 g glycine), 150 ml sucrose syrup, lemon extract (for flavor improvement), preservative, and water to a volume of 300 ml. The pH of the L-arginine and glycine solutions differed slightly, ~3.0 for the L-arginine drink and ~1.5 for the glycine drink. The subjects were instructed to drink 75 ml of amino acid solution four times a day, accompanied by a meal when possible. Use of a daily L-arginine dose of 30 g for 8 days is based on previous studies in healthy humans and in patients and is described as a safe dose (2, 6, 13).
Stationary esophageal manometry. Stationary manometric recordings were obtained with a six-channel water-perfused catheter (OD 4.8 mm) with a Dent sleeve. After introduction through the nose, the catheter was positioned with three side holes in the esophagus at 5, 10, and 15 cm above the LES, with the sleeve in the LES, and with one side hole in the fundus of the stomach. The catheter was connected to a low-compliance pneumohydraulic perfusion system and perfused with deionized water at a constant rate of 0.3 ml/min. Pressures were measured by external transducers (DPT-200; Medisize, Hillegom, The Netherlands) and stored in a digital portable data logger (MMS, Enschede, The Netherlands) using a sample frequency of 8 Hz. A separate solid-state catheter (OD 2 mm; Braun Medical, Oss, The Netherlands) was used to record pharyngeal pressure peaks caused by swallowing. Both catheters were fixed to the nose.
Ambulatory esophageal monitoring. Ambulatory manometric recordings were obtained for 24 h, using a catheter with two solid-state pressure transducers (OD 2.0 mm; P.P.G. Hellige, Best, The Netherlands). Esophageal pH was recorded with a polyurethane assembly containing 5 ion-sensitive field effect transistor pH transducers at 3-cm intervals (OD 2.7 mm; Sentron, Roden, The Netherlands) (38). After introduction through the nose, the pressure transducers were positioned at 5 and 15 cm proximal to the upper border of the LES; the pH transducers were at 3, 6, 9, 12, and 15 cm proximal to the LES. Both catheters were fixed to the nose and connected to a portable data logger (MMS). Pressure signals were sampled at a rate of 4 Hz and pH signals at a rate of 2 Hz. The data logger contained buttons for marking consumption of meals and beverages and recumbent time. Subjects were also instructed to record these times of consumption and to note times spent in the supine position on a diary form. Intake of acidic food and beverages (pH <5) and extreme physical activity were to be avoided.
Gallbladder ultrasonography. Gallbladder volumes were measured by real-time ultrasonography (Scanner 250, 3.5/5 MHz convex transducer; Pie Medical, Maastricht, The Netherlands). Subcostal sonographic images were obtained in duplicate with the subjects supine. Longitudinal and transverse images of the gallbladder at its largest dimensions were obtained. Gallbladder volume was calculated on-line according to the sum-of-cylinders method (8). The fasting volumes on days 1 and 7 and the residual volume on day 7 were calculated. The ejection volume was calculated as the difference between fasting and residual gallbladder volume.
Blood analysis.
Plasma levels of sodium, potassium, chloride, bicarbonate, and urea
were measured. Plasma levels of free amino acids (arginine, glycine,
citrulline, and ornithine) were measured by ion-exchange chromatography
(Biotronic LC 5001). For free amino acid measurement, a volume of
norleucine-containing sulfosalicylic acid, equal to 3.3% of the plasma
volume, was added to the blood plasma and a thymol crystal was
added. The blood samples were frozen (20°C) to
await further analysis.
Urine analysis. Urine was collected for 24 h for measurement of creatinine and nitrite/nitrate excretion. Excretion of nitrite and nitrate, the oxidation products of NO, is indicated as NOx. NOx concentration was determined by automated flow analysis (12). Urinary NOx excretion (in mmol NOx/mol urinary creatinine) was checked for inaccuracy of urine collection by the subjects.
Analysis of pressure recordings.
Recordings obtained during stationary esophageal manometry were
analyzed for basal LESP and relaxations of the LES, using computer
algorithms. The algorithms first calculated the LESP with respect to
fundic pressure (as kPa above fundic pressure). This was done by
subtracting the fundic pressure signal from the LESP signal derived
from the sleeve device. Second, relaxations of the LES were detected
automatically, using the following criteria: relaxations should occur
at a minimum rate of 0.4 kPa in 3 s, to a pressure of <0.1 kPa above
gastric fundic pressure for at least 1 s. The LES relaxations were then
classified visually as "swallow-induced" or
"spontaneous/transient". LES relaxations were considered as
transient when one of the following two criteria, according to Mittal
et al. (23), was met: 1) absence of
a pharyngeal swallow signal for 4 s before to 2 s after the onset of
LES relaxation with a decrease rate for LESP of >0.13 kPa/s to an
LESP of 0.1 kPa within 10 s; or 2)
relaxation to
0.1 kPa for >10 s, irrespective of the timing of LES
relaxation in response to swallowing; LES relaxations associated with
multiple rapid swallows were excluded. Mean basal LESP was calculated
for 15-min periods for each subject. This was done after exclusion of
LES relaxations and after contractions, using intervals beginning at
the start of LES relaxation and ending 8 s after the end of LES
relaxation. The percentages of peristaltic, simultaneous, and
nontransmitted contractions were evaluated for the esophageal pressure
signals from both stationary and ambulatory esophageal manometry. The
peristaltic contractions were analyzed for mean amplitude, duration,
and propagation velocity. Stationary esophageal manometry recordings
were analyzed for dry and wet swallows separately. Ambulatory manometry
recordings were analyzed separately for times in the upright and the
supine positions. Meal and beverage periods were excluded from the
analysis.
Analysis of esophageal pH recordings. Recordings of esophageal pH were analyzed automatically by means of locally developed computer software (34). Reflux parameters from the five channels (percentage of time with pH <4, number of episodes with pH <4, number of episodes with pH <4 lasting 5 min or more, and mean duration of episodes) were calculated separately for periods of upright and supine positions and for the total recording time. The values for numbers of reflux episodes were normalized to periods of 16 h (upright position), 8 h (supine position), and 24 h (total recording time).
Statistical analysis. Differences in LESP for the entire recording period were analyzed using ANOVA for repeated measures. When differences were significant, further analysis with contrast methods was used to analyze in detail the time at which a change in LESP occurred. Missing values in LESP were replaced by the series mean of that 15-min period to make paired analysis possible. TLESR frequency and duration were compared by Student's t-test for paired data. Differences in esophageal pressure and pH parameters were analyzed using Student's t-test for paired data (for normally distributed parameters) or Wilcoxon's test (for nonparametric parameters). Differences between L-arginine and glycine in pH parameters at all esophageal levels were analyzed using the ANOVA for repeated measures. Gallbladder volumes were compared by means of Student's t-test for paired data. Values for blood and urine parameters were analyzed for differences using Student's t-test for paired data. Statistical significance was defined as a two-tailed P < 0.05.
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RESULTS |
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Data obtained from one of the subjects had to be excluded from analysis, because his plasma L-arginine levels showed no increase after 6 days of L-arginine intake. His intake of L-arginine was considered to be unreliable. Stationary esophageal manometry data could be analyzed for only seven subjects, as data for two subjects were lost through computer failure. Ambulatory manometry data were analyzed for eight subjects, due to early ending of esophageal manometry in one subject. Reflux data were not complete for all levels in all subjects, because of technical failures. Plasma levels were missing for one subject on day 7 of L-arginine. Urinary data during glycine intake were missing for one subject.
LES. During the first 60 min postprandial, the mean LESP (n = 7) for L-arginine and glycine intake differed (Fig. 2; P < 0.02), with a mean LESP of 2.16 ± 0.06 vs. 2.65 ± 1.20 kPa for L-arginine vs. glycine, respectively. During the glycine period, a significant increase in LESP was observed starting at ~75 min postprandial (P < 0.01), reaching a plateau of 3.80 ± 0.41 kPa at 135 min postprandial. There was no such late postprandial increase of LESP during L-arginine intake, and LESP remained at the early postprandial levels (LESP of 2.14 ± 0.10 kPa at 135 min postprandial). The frequency of TLESRs did not differ significantly for L-arginine and for glycine, being 12.0 ± 4.6 and 10.7 ± 4.9 TLESRs, respectively, for 3 h postprandial. One subject had an extremely high frequency of 33 and 36 TLESRs (in 3 h) after L-arginine and glycine, respectively, and two subjects showed no TLESRs. The duration of TLESRs was significantly prolonged, by ~16%, after L-arginine intake; durations were 18.0 ± 1.3 and 15.1 ± 1.0 s for L-arginine and glycine, respectively (P < 0.02; n = 5).
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Esophageal body. Amplitude, duration, and propagation velocity of esophageal contractions did not differ significantly between L-arginine and glycine intake. This was found for both wet and dry swallows, during stationary esophageal manometry and ambulatory esophageal manometry, even after separate analysis for the upright or supine position and for preprandial periods. In addition, no differences were found in the type of contractions, i.e., peristaltic, simultaneous, or nontransmitted contractions.
Gastroesophageal reflux. Table 1 shows reflux parameters (%reflux time and number of reflux episodes) for different levels in the esophagus, after both L-arginine and glycine. These reflux parameters showed a gradual decrease from the distal to the proximal recording site, following a linear pattern, both for L-arginine and glycine. The mean duration of reflux episodes was the same for all esophageal levels (not shown in Table 1). The values for the 24-h reflux parameters were not significantly different between L-arginine and glycine intake, whether for the upright and supine periods measured separately or for the total 24-h period. However, the percentage of reflux time as well as the mean duration of reflux episodes during the supine period after L-arginine tended to be higher than after glycine at all esophageal levels (P = 0.1). Statistical significance was not reached, though, due to the low number of episodes in the supine period.
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Gallbladder. Figure 3 shows the fasting and residual gallbladder volumes for L-arginine vs. glycine (n = 8). One subject had to be excluded from the analysis because of a lack of postprandial gallbladder contraction, after both L-arginine and glycine. On day 1 (before intake of L-arginine and glycine) basal fasting gallbladder volumes were 18.9 ± 1.7 and 19.0 ± 1.9 ml, respectively (not significant). After 6 days of intake there was a significant difference in fasting gallbladder volume between L-arginine and glycine (22.9 ± 1.7 and 18.5 ± 1.5 ml, respectively; P < 0.05). The fasting gallbladder volume after glycine was not significantly different from the fasting gallbladder volume before the start of glycine intake (as was also shown in the pilot study). The fasting gallbladder volume after L-arginine was significantly increased, by 21.9 ± 1.7%, compared with the fasting gallbladder volume before the start of L-arginine intake (P < 0.05). The residual volumes after the meal on day 7 were also different, being 7.7 ± 1.6 vs. 3.4 ± 0.9 ml for L-arginine vs. glycine, respectively (P < 0.05). The gallbladder emptying percentage after L-arginine was smaller than after glycine (67.4 ± 6.2% and 81.7 ± 4.3%, respectively; P < 0.05). However, due to the simultaneous increase of both fasting and residual volume after L-arginine intake, the absolute ejection volume was not significantly different, being 15.2 ± 1.6 vs. 15.0 ± 1.5 ml for L-arginine vs. glycine, respectively.
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Blood chemistry. Table 2 shows the plasma levels of free amino acids (arginine, glycine, citrulline, ornithine), urea and electrolytes (sodium, potassium, chloride, and bicarbonate) before L-arginine and glycine (day 1), respectively, and after 6 days of intake (day 7). Intake of L-arginine significantly changed the plasma levels of arginine, ornithine, urea, chloride, and bicarbonate (P < 0.005). Ornithine and urea are metabolic products of L-arginine, which explains the observed increases of plasma ornithine and urea after L-arginine intake. Glycine intake changed the plasma levels of glycine, chloride and bicarbonate significantly (P < 0.005). As expected, glycine intake did not significantly change the plasma levels of arginine. No differences were observed between the values for blood parameters before L-arginine and glycine (day 1) (P > 0.05).
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Urine chemistry. NOx excretion after 7 days of L-arginine intake was significantly higher [63.7 ± 8.3 mmol/mol creatinine (mean ± SE)] than after 7 days of glycine intake (54.5 ± 6.8 mmol/mol creatinine) (P < 0.05) (Fig. 4). No differences in 24-h urinary creatinine excretion were observed between L-arginine and glycine (17.8 ± 1.6 and 17.4 ± 0.8 mmol/24 h, respectively; P > 0.05).
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DISCUSSION |
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This study provides evidence that after several days of an increased daily intake of L-arginine NO production increases, as was shown by enhanced urinary NOx excretion. Esophageal motility was not affected, but LES tone and relaxation were altered. The postprandial LESP was lowered, the normal late postprandial LESP increase was suppressed, and the duration of TLESRs was prolonged, while the occurrence of reflux was not affected significantly. Long-term oral L-arginine also increased fasting and residual gallbladder volume. These effects of L-arginine on the LES and the gallbladder may be mediated by NO. It is not clear why oral L-arginine selectively affected only the LES and the gallbladder, without an effect on esophagus and blood pressure. It is possible that differences in sensitivity to NO changes between the organs exist or that organ-specific long-term adaptation occurs.
Urinary NOx excretion has been
shown to be a good parameter for endogenous NO synthesis from
L-arginine (5, 18),
although the measurement of endogenously generated
NOx excretion may be confounded by
several factors. The most important of these factors is dietary
NOx intake. We therefore regulated
the food intake for each subject by using a food diary and standardized
dinners, starting one day before urine collection. A second source for NOx, NO inhaled via tobacco smoke,
could be eliminated since all subjects were nonsmokers. The route of
administration of L-arginine also affects NOx biosynthesis.
Oral L-arginine administration results in a more extensive transfer of labeled nitrogen atoms of
L-arginine to urinary
NOx than does intravenous
administration (5). Also, continuous infusion of low-dose
[15N]arginine
intragastrically results in a greater recovery of
15NO3
than does a large oral bolus of labeled L-arginine (5, 18). We therefore
assumed that long-term repeated oral administration of
L-arginine, as used in our
study, would yield maximal NO synthesis from
L-arginine and be a better model of the physiological situation than would acute high-dose
L-arginine intravenously. The
difference in NOx excretion with
long-term oral L-arginine and
that with glycine was small but significant, indicating a small
increase in endogenous NOx
excretion from
L-arginine-derived NO
production. However, the results we obtained do not allow us to
conclude whether NOx is derived
from the oxidation of NO produced by constitutive NOS (especially
neuronal NOS associated with NANC innervation) or of NO produced by
inducible NOS, which is expressed in many cell types after immune
stimulation. Because all subjects were in good health during the
experiment, we assumed that the increased urinary
NOx excretion seen with
L-arginine reflects activation
of the constitutive
L-arginine-NO pathway.
We found an effect of long-term oral L-arginine on LES and the gallbladder. The early postprandial LESP with L-arginine was lowered, and the physiological late postprandial LESP increase (33) was suppressed. The effect on early postprandial LESP corresponds with the decreased basal LESP seen in humans after administration of the NO donor molsidomine (39). Our findings are also consistent with the increase in LES resting pressure seen after administration of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) to humans, an increase which can be reversed by intravenous L-arginine (16). As far as we know, no studies in humans on the effect of L-arginine alone, administered orally or intravenously, have yet been reported. Studies of the opossum revealed no effect of L-arginine alone on resting LES tone in vitro (26, 40) and on LESP in vivo (29). The possibility of a role of the L-arginine-NO pathway in the regulation of human LESP is supported by the fact that organic nitrates, such as nitroglycerin and isosorbide trinitrate, were used in the treatment of achalasia patients to lower the LESP (10), even before it was known that these nitrates act through NO pathways. No NOS was detectable in the myenteric plexus of the LES in these patients, possibly explaining the impaired function of the LES (22).
Our observation that the late postprandial increase in LESP does not occur after L-arginine ingestion may be explained in various ways. First, a direct increase in inhibitory activity of NANC nerves with NO as neurotransmitter might be involved, as described above. Another explanation may be that the postprandial period is prolonged by L-arginine as a result of delayed gastric emptying. The physiological late postprandial increase in LESP (33) may therefore be delayed. Delayed gastric emptying caused by glyceryl trinitrate and by intravenous or intragastric L-arginine has been reported in humans (17, 35) and dogs (27). In the latter, the effect of L-arginine was ascribed primarily to suppression of gastric contractions (27). CCK may be involved in this response, since it delays gastric emptying and the gastric response to CCK is mediated vagally by the NO system (19). L-Arginine increases the release of several gastrointestinal hormones, such as gastrin, glucagon, and somatostatin (17, 36, 37), the action of which might also explain the effect of L-arginine on postprandial LESP. For example, gastrin has been reported to have an excitatory effect on LES, and somatostatin and glucagon have an inhibitory effect (30). However, meal-induced glucagon and somatostatin release is not increased by intravenous L-arginine (9, 17), and serum gastrin is not affected by either orally or intragastrically administered L-arginine (35, 36). It therefore seems unlikely that the postprandial effects of oral L-arginine that we observed were mediated through the release of gastrin, somatostatin, or glucagon.
TLESR frequency was not affected by L-arginine, but the mean duration of TLESRs was significantly prolonged after L-arginine in our human study. The mechanism determining meal-induced TLESRs is suggested to involve CCK, with NO as the neurotransmitter released at the postganglionic site in the vagal pathway (3, 23). Frequency of TLESRs triggered by gastric distension is shown to be lowered by the NOS inhibitor NG-nitro-L-arginine methyl ester in dogs (3). In our study, L-arginine had no effect on TLESR frequency. A possible explanation may be that NO increase (by L-arginine) and NO decrease (by NOS inhibitors) do not necessarily have an equal opposite effect. Second, the increase in NO in our study was only small. The mechanism controlling TLESR duration is not known, although the CCK-A receptor antagonist devazepide reduced the duration of TLESRs in dogs, while NOS inhibition was not effective (3). On the basis of our findings, we suggest that the L-arginine-NO pathway may also be involved in the duration of TLESRs. TLESRs are now recognized as the most important mechanism of gastroesophageal reflux. TLESRs associated with reflux have been shown to be significantly longer than those not associated with reflux (33). In our study we did not observe an effect of L-arginine on the frequency of reflux episodes. However, the duration of reflux episodes during the supine period tended to be increased after L-arginine. As the decrease in reflux occurrence from distal to proximal in the esophagus was equal for L-arginine and glycine, this might indicate that the acid clearance was not affected by L-arginine. Moreover, esophageal motility as an important mechanism for acid clearance was not changed after L-arginine in our study. Longer TLESR duration and longer duration of reflux episodes at all esophageal levels might indicate that the volume of refluxate is increased. However, since TLESRs and reflux were not measured simultaneously in our study, we need to be very careful in associating the two, since that requires further investigation.
We found no effects of L-arginine on tubular esophageal motility, i.e., amplitude, duration, and propagation velocity of contractions, although effects in response to NO donors and NOS inhibitors have been reported for the amplitude and propagation velocity of esophageal contractions, especially in the distal esophagus (1, 14, 16, 39, 41). L-Arginine alone has no effect on esophageal peristalsis either in vitro or in vivo in experimental animals (26, 41). The absence of any effect of L-arginine on these parameters in our study may be explained by the fact that the increases in NO synthesis were probably slight compared with the changes in NO levels in studies using NO donors or NOS inhibitors.
In addition to an effect of L-arginine on the LES, we found an increase in fasting and residual gallbladder volume and a reduction in gallbladder emptying after L-arginine. Similar results have been described for intravenous L-arginine (9) and for the NO donor glyceryl trinitrate (11). This suggests that the effect of L-arginine in our study may, as hypothesized, have been NO mediated and that NO in vivo might have an inhibitory influence on gallbladder motility. It is not known whether the effect of L-arginine on gallbladder volume occurs through stimulation of NO synthesis in NANC nerves or in the gallbladder smooth muscle cells themselves. NOS-positive neurons have been identified in both the mucosa and neurons innervating the muscularis in humans (31). The effect of L-arginine on gallbladder emptying may also be explained by an inhibition of CCK-induced gallbladder contractions, since the NOS inhibitor L-NMMA reduces fasting gallbladder volume and augments CCK- or meal-induced gallbladder emptying in vivo in humans (15). In accordance with this, NO completely abolishes CCK-induced gallbladder contraction in vitro (31).
In conclusion, long-term oral L-arginine affects the LESP by lowering the basal postprandial LESP and by suppressing the physiological late postprandial LESP increase. Furthermore, fasting and residual gallbladder volumes are increased after L-arginine ingestion. These effects of L-arginine might both be mediated through the L-arginine-NO pathway. Further studies are needed to assess the role of CCK and other gastrointestinal hormones in the L-arginine-induced changes in LES and gallbladder motility.
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ACKNOWLEDGEMENTS |
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We acknowledge the Department of Human and Animal Physiology of the Agricultural University Wageningen (Wageningen, The Netherlands) for analysis of plasma levels of free amino acids.
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FOOTNOTES |
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This study was supported by The Netherlands Organization for Scientific Research, Council for Medical and Health Research (NWO-GMW) Grant 900-522-140.
A portion of this study was presented at the annual meeting of the American Gastroenterological Association in San Diego, CA, in May 1995, and has been published previously in abstract form (Gastroenterology 108: A642, 1995).
Address for reprint requests: Y. C. Luiking, Univ. Hospital Utrecht, Dept. of Experimental Surgery, HP G.04.228, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
Received 11 July 1997; accepted in final form 29 January 1998.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anand, N.,
and
W. G. Paterson.
Role of nitric oxide in esophageal peristalsis.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G123-G131,
1994
2.
Barbul, A.,
S. A. Lazarou,
D. T. Efron,
H. L. Wasserkrug,
and
G. Efron.
Arginine enhances wound healing and lymphocyte immune responses in humans.
Surgery
108:
331-337,
1990[Medline].
3.
Boulant, J.,
J. Fioramonti,
M. Dapoigny,
G. Bommelaer,
and
L. Bueno.
Cholecystokinin and nitric oxide in transient lower esophageal sphincter relaxation to gastric distention in dogs.
Gastroenterology
107:
1059-1066,
1994[Medline].
4.
Brookes, S. J. H.,
B. N. Chen,
W. M. Hodgson,
and
M. Costa.
Characterization of excitatory and inhibitory motor neurons to guinea pig lower esophageal sphincter.
Gastroenterology
111:
108-117,
1996[Medline].
5.
Castillo, L.,
T. C. DeRojas,
T. E. Chapman,
J. Vogt,
J. F. Burke,
S. R. Tannenbaum,
and
V. R. Young.
Splanchnic metabolism of dietary arginine in relation to nitric oxide synthesis in normal adult man.
Proc. Natl. Acad. Sci. USA
90:
193-197,
1993[Abstract].
6.
Daly, J. M.,
J. Reynolds,
A. Thom,
L. Kinsley,
M. Dietrick-Gallagher,
J. Shou,
and
B. Ruggieri.
Immune and metabolic effects of arginine in the surgical patient.
Ann. Surg.
208:
512-523,
1988[Medline].
7.
Dent, J.,
W. J. Dodds,
R. H. Friedman,
T. Sekiguchi,
W. J. Hogan,
R. C. Arndorfer,
and
D. J. Petrie.
Mechanism of gastroesophageal reflux in recumbent asymptomatic human subjects.
J. Clin. Invest.
65:
256-267,
1980[Medline].
8.
Everson, G. Y.,
D. Z. Braverman,
M. L. Johnson,
and
F. Kern.
A critical evaluation of real-time ultrasonography for the study of gallbladder volume and contraction.
Gastroenterology
79:
40-46,
1980[Medline].
9.
Fiorucci, S.,
E. Distrutti,
A. Quintieri,
L. Sarpi,
Z. Spirchez,
N. Gulla,
and
A. Morelli.
L-Arginine/nitric oxide pathway modulates gastric motility and gallbladder emptying induced by erythromycin and liquid meal in humans.
Dig. Dis. Sci.
40:
1365-1371,
1995[Medline].
10.
Gelfond, M.,
P. Rozen,
and
T. Gilat.
Isosorbide dinitrate and nifedipine treatment of achalasia: a clinical manometric and radionuclide evaluation.
Gastroenterology
83:
963-969,
1982[Medline].
11.
Greaves, R. R. S. H.,
J. H. Miller,
L. J. D. O'Donnell,
A. McLean,
and
M. J. G. Farthing.
Glyceryl trinitrate reduces gallbladder emptying in healthy subjects (Abstract).
Gastroenterology
110:
A670,
1996.
12.
Green, L. C.,
D. A. Wagner,
J. Glogowski,
P. L. Skipper,
J. S. Wishnok,
and
S. R. Tannenbaum.
Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.
Anal. Biochem.
126:
131-138,
1982[Medline].
13.
Kirk, S. J.,
M. Hurson,
M. C. Regan,
D. R. Holt,
H. L. Wasserkrug,
and
A. Barbul.
Arginine stimulates wound healing and immune function in elderly human beings.
Surgery
114:
155-160,
1993[Medline].
14.
Knudsen, M. A.,
O. Frobert,
and
A. Tøttrup.
The role of the L-arginine-nitric oxide pathway for peristalsis in the opossum oesophageal body.
Scand. J. Gastroenterol.
29:
1083-1087,
1994[Medline].
15.
Konturek, J. W.,
N. Kwiecien,
E. Sito,
S. J. Konturek,
and
W. Domschke.
Physiological role of nitric oxide in gallbladder contractions in man (Abstract).
Gastroenterology
108:
A422,
1995.
16.
Konturek, J. W.,
M. Maczka,
P. Thor,
A. Gabryelewicz,
R. Stoll,
and
W. Domschke.
Endogenous nitric oxide in the control of esophageal motility in humans (Abstract).
Gastroenterology
106:
A526,
1994.
17.
Konturek, J. W.,
P. Thor,
and
W. Domschke.
Effect of nitric oxide on antral motility and gastric emptying in humans.
Eur. J. Gastroenterol. Hepatol.
7:
97-102,
1995[Medline].
18.
Leaf, C. D.,
J. S. Wishnok,
and
S. R. Tannenbaum.
L-Arginine is a precursor for nitrate biosynthesis in humans.
Biochem. Biophys. Res. Commun.
163:
1032-1037,
1989[Medline].
19.
Martinez, V.,
M. Jiminez,
E. Goñalons,
and
P. Vergara.
Mechanism of action of CCK in avian gastroduodenal motility: evidence for nitric oxide involvement.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G842-G850,
1993
20.
McCallum, R. W.,
B. Kuljian,
R. H. Holloway,
and
J. H. Walsh.
Effect of intragastric amino acids on lower esophageal sphincter pressure and serum gastrin in man.
Am. J. Gastroenterol.
81:
168-171,
1986[Medline].
21.
McKirdy, M. L.,
H. C. McKirdy,
and
C. D. Johnson.
Non-adrenergic non-cholinergic inhibitory innervation shown by electrical field stimulation of isolated strips of human gallbladder muscle.
Gut
35:
412-416,
1994[Abstract].
22.
Mearin, F.,
M. Mourelle,
F. Guarner,
A. Salas,
V. Riveros-Moreno,
S. Moncada,
and
J. R. Malagelada.
Patients with achalasia lack nitric oxide synthase in the gastro-oesophageal junction.
Eur. J. Clin. Invest.
23:
724-728,
1993[Medline].
23.
Mittal, R. K.,
R. H. Holloway,
R. Penagini,
L. A. Blackshaw,
and
J. Dent.
Transient lower esophageal sphincter relaxation.
Gastroenterology
109:
601-610,
1995[Medline].
24.
Moncada, S.,
and
A. Higgs.
The L-arginine-nitric oxide pathway.
N. Engl. J. Med.
329:
2002-2012,
1993
25.
Mourelle, M.,
F. Guarner,
X. Molero,
S. Moncada,
and
J. R. Malagelada.
Regulation of gallbladder motility by the arginine nitric oxide pathway in guinea pigs.
Gut
34:
911-915,
1993[Abstract].
26.
Murray, J.,
C. Du,
A. Ledlow,
J. N. Bates,
and
J. L. Conklin.
Nitric oxide: mediator of nonadrenergic noncholinergic responses of opossum esophageal muscle.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G401-G406,
1991
27.
Orihata, M.,
and
S. K. Sarna.
Inhibition of nitric oxide synthase delays gastric emptying of solid meals.
J. Pharmacol. Exp. Ther.
271:
660-670,
1994[Abstract].
28.
Palmer, R. M. J.,
A. G. Ferrige,
and
S. Moncada.
Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor.
Nature
327:
524-526,
1987[Medline].
29.
Paterson, W. G.,
M. A. B. Anderson,
and
N. Anand.
Pharmacological characterization of lower esophageal sphincter relaxation induced by swallowing, vagal efferent nerve stimulation, and esophageal distention.
Can. J. Physiol. Pharmacol.
70:
1011-1015,
1992[Medline].
30.
Reynolds, J. C.
Anatomy, abnormalities, and physiology of the esophagus. Part 2: Physiology.
In: Bockus Gastroenterology, edited by W. S. Haubrich,
F. Schaffner,
and J. E. Berk. Philadelphia, PA: Saunders, 1995, p. 411-417.
31.
Salomons, H. S.,
W. LaMorte,
A. Burke,
A. Sangupta,
G. D. Offner,
and
N. H. Afdhal.
Nitric oxide is a potent inhibitor of gallbladder contractility (Abstract).
Hepatology
20:
142A,
1994.
32.
Sanders, K. M.,
and
S. M. Ward.
Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission.
Am. J. Physiol.
262 (Gastrointest. Liver Physiol. 25):
G379-G392,
1992
33.
Schoeman, M. N.,
M. D. Tippett,
L. M. A. Akkermans,
J. Dent,
and
R. H. Holloway.
Mechanisms of gastroesophageal reflux in ambulant healthy human subjects.
Gastroenterology
108:
83-91,
1995[Medline].
34.
Smout, A. J. P. M.,
M. Breedijk,
C. van der Zouw,
and
L. M. A. Akkermans.
Physiological gastroesophageal reflux and esophageal motor activity studied with a new system for 24-hour recording and automated analysis.
Dig. Dis. Sci.
34:
372-378,
1989[Medline].
35.
Taylor, I. L.,
W. J. Byrne,
D. L. Christie,
M. E. Ament,
and
J. H. Walsh.
Effect of individual L-amino acids on gastric acid secretion and serum gastrin and pancreatic polypeptide release in humans.
Gastroenterology
83:
273-278,
1982[Medline].
36.
Vinik, A. I.,
W. J. Kalk,
D. M. Dent,
G. Barbezat,
B. J. Grant,
and
S. Bank.
Stimuli for heptadecapeptide gastrin release: a comparison of oral and intravenous arginine-monochloride and oxo in normal, vagotomized and antrectomized patients.
Scand. J. Gastroenterol.
10:
97-100,
1975[Medline].
37.
Weir, G. C.,
E. Samols,
S. Loo,
Y. C. Patel,
and
K. H. Gabbay.
Somatostatin and pancreatic polypeptide secretion. Effects of glucagon, insulin, and arginine.
Diabetes
28:
35-40,
1979[Abstract].
38.
Weusten, B. L. A. M.,
L. M. A. Akkermans,
G. P. van Berge Henegouwen,
and
A. J. P. M. Smout.
Spatiotemporal characteristics of physiological gastroesophageal reflux.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G357-G362,
1994
39.
Willis, S.,
H. D. Allescher,
B. Stotschus,
V. Schusdziarra,
M. Classen,
and
V. Schumpelick.
Double blind placebo controlled study on the effect of the nitric oxide donor molsidomin and the 5-HT3 antagonist ondansetron on human esophageal motility.
Z. Gastroenterol.
32:
632-636,
1994[Medline].
40.
Yamato, S.,
J. K. Saha,
and
R. K. Goyal.
Role of nitric oxide in lower esophageal sphincter relaxation to swallowing.
Life Sci.
50:
1263-1272,
1992[Medline].
41.
Yamato, S.,
S. J. Spechler,
and
R. K. Goyal.
Role of nitric oxide in esophageal peristalsis in the opossum.
Gastroenterology
103:
197-204,
1992[Medline].