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Neural control of the lower esophageal sphincter in the cat: Studies on the excitatory pathways to the lower esophageal sphincter
GASTROENTEROLOGY
1982;82:680-8
Neural Control of the Lower Esophageal Sphincter in the Cat: Studies on the Excitatory Pathways to the Lower Esophageal Sphincter JOSE BEHAR, MORRIS KERSTEIN, and PIER0 BIANCANI The Department of Medicine, Rhode Island Hospital and Brown University, Providence, Rhode Island, and the Department of Surgery, Tulane University, New Orleans, Louisiana
Lower esophageal sphincter pressures were measured with a 6-lumen catheter anchored at the high pressure point by a metallic pin. Electrical stimulation (5-40 Hz) of both central ends of the cervical vagus caused contraction of the lower eSOphQgeQ1 sphincter, which was antagonized by section of the spinal cord above the brachial plexus, by tetrodotoxin, by a combination of hexamethonium and atropine, by phentolamine, and by catecholamine depletion with reserpine. Stimulation (5-40 Hz] of peripheral or central ends of the left sphmchnic nerves also caused contraction of the lower esophageal sphincter. This sphincter response was antagonized by tetradotoxin, a combination of hexamethonium and atropine, phentolamine, and catecholamine depletion With reserpine. It was not affected by atropine alone or by propranolol. Lower esophageal sphincter relaxation was not observed even after the contraction was blocked with phQrmQcologic antagonists. Stimulation (5-40 Hz) of the peripheral end of the periarterial nerves of the left gastric artery also caused contraction of the sphincter and was antagonized only by tetrodotoxin and phentolamine. Atropine and hexamethonium did not affect this sphincter response. Extrinsic denervation of the lower esophageal sphincter by sectioning of the cervical vagus nerves, the spinal cord, the left sphmchnic nerves, or the left gastric artery did not affect basal sphincter pressures. BQSQ~ sphincter pressure was not affected by atropine alone, by Q combination of hexamethonium and atropine, or by Received June 29, 1981. Accepted November 12, 1981. Address requests for reprints to: Dr. Jose Behar, APC Building, Room 421, Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02902. The authors thank Carol Marin, Susan Field, and Judy Johnson for the laboratory assistance, and Lorie Klemer for her secretarial help. 0 1982 by the American Gastroenterological 0016-5085/82/040680-09$02.50
Association
propranolol. There was, however, a 25%-28% reduction with tetrodotoxin and phentolamine. These findings suggest the existence of an excitatory reflex arc with an afferent vagaJ and efferent sympathetic pathway. It consists of pregcmgbonic cholinergic fibers and postganglionic adrenergic neurons synapsing with Q-receptors at the muscle fiber. These adrenergic neurons may contribute to the genesis of bQSQ1 lower esophageal sphincter tone, or to its regulation in balance with tonic neural inhibitory influences. Lower esophageal sphincter (LES) closure is determined by tension of the circular muscle (1,2). The mechanism by which sphincter closure is maintained, however, has not been elucidated. In vitro studies using circular muscle strips from the LES of the opossum and cat have shown that at the gastroesophageal junction there is a specialized circular muscle layer with steeper length-tension curves than those from adjacent areas (3,4). In the opossum, in vivo basal sphincter pressures are only slightly or not at all affected by any of the neural antagonists, including tetrodotoxin (TTX) (5). Other in vivo studies (7-g), however, have presented indirect evidence that neural mechanisms may contribute to the genesis of sphincter tone. The magnitude of the neural contribution is different in various animal species. In the opossum, LES innervation does not appear to play any role in the genesis of basal tone (5); while in the cat, we have shown a reduction of 25%-28% in LES pressures after the administration of TTX or of an a-adrenergic antagonist such as phentolamine (6). In conscious humans, atropine and probanthine decrease basal sphincter pressures significantly (7,B). Similarly, the dog LES is affected by atropine (9). Excitatory pathways to the LES have been shown in both the opossum and the cat by using electrical stimulation of extrinsic nerves (6,10,11). Stimulation
April
1982
of afferent vagal fibers and of periarterial sympathetic fibers of the left gastric artery (LGA) caused LES contraction. The purposes of the present study were: to investigate the excitatory neural pathways to the cat LES, to determine the nature of the neural fibers present, and to assess their contribution to basal sphincter tone.
Materials and Methods These studies were performed in 86 adult cats of either sex weighing 3-4 kg. After fasting for 16 h, the animals were anesthetized intramuscularly with ketamine hydrochloride (30 mg/kg body wt) and were kept lightly anesthetized with maintenance doses of 5-10 mgikg throughout the experiment. Controlled ventilation was maintained in all animals with a respirator (Harvard Apparatus Co., South Natick, Mass.) via a tracheostomy tube. All animals were supine when studied. Normal saline with 5% dextrose was slowly infused through an iv. cannula. Arterial blood pressure was recorded by cannulating the femoral artery with a catheter connected to a Statham pressure transducer. Esophageal, LES, and gastric pressures were recorded with a low-compliance system using a constant hydraulic infusion pump. A water-filled, g-lumen catheter assembly was perfused at the rate of 0.3 mllmin, connected to a Statham pressure transducer and a 12-channel Grass polygraph. The rate of pressure increase of this system with occlusion of the manometric probe (>lOO mmHg) was greater than the observed changes in LES pressure. The catheter assembly consisted of six polyvinyl tubes (OD of each = 1.6 mm), glued on the anterior and posterior aspects of a hollow polyvinyl tube (OD = 3.4 mm). The dimensions of the entire assembly were 6.6 mm in its longest and 3.4 mm in its shortest diameter. The catheter was introduced into the stomach through the mouth. The LES was located with two gradual pullthroughs by using the four side openings, 1 mm apart. Gastric pressure was measured by the side opening 1 cm distal, and esophageal pressure was measured with the side opening 2 cm proximal to the four central openings. After an abdominal midline incision, the gastroesophageal junction was exposed, and the probe was anchored at this level by inserting a thin, metallic pin through the LES wall and lateral side of the hollow tubing. The pin was covered with small corks at both ends to avoid damage to the adjacent organs. The pin-anchored catheter assembly permitted evaluation of the LES pressures at the same level and radial orientation throughout the experiment. Lower esophageal sphincter pressure throughout the experiment was stable. After the abdominal incision was covered with wet gauze, basal sphincter pressure was allowed to stabilize for 30 min before beginning the study. The LES was defined as a zone of high pressure that relaxed during electrical stimulation of the peripheral end of the vagus nerve or during distention of a small rubber balloon placed in midesophagus. Placement of the metallic pin through the LES did not affect LES function (11).Water infused
NEURAL CONTROL OF THE CAT LES
681
into the stomach through the six side openings was allowed to drain by gravity through the hollow core tube. This continuous drainage prevented distention of the stomach.
Experimental
Studies
The vagus nerves were identified and carefully exposed at the level of the neck in all studies. For those studies involving the splanchnic nerves, the left celiac ganglia and its branches were identified and carefully dissected at the origin of the celiac artery. The left splanchnit nerves were secured by tying two silk sutures, and they were then sectioned into peripheral ends going into the ganglia and central ends leading back to the sympathetic chain and spinal cord. The spinal cord was severed above the level of the brachial plexus for those studies involving spinal cord section. The completeness of the section was determined by finding that reflexes in all four extremities were abolished. For studies involving the LGA, the artery was identified and isolated with its periarterial connective tissue and plexus. It was secured by tying two silk sutures around it, and then severed into a peripheral end leading to the gastroesophageal junction and a central end to the porta hepatis. Lower esophageal sphincter pressures were monitored under the following conditions: [a) electrical stimulation of both central ends of the cervical vagus nerve before and after pharmacologic antagonists; (b) electrical stimulation of central and peripheral ends of both left greater and lesser splanchnic nerves, before and after treatment with pharmacologic antagonists; (c) before and after spinal cord section in basal conditions and during electrical stimulation; (d) electrical stimulation of the periarterial sympathetic nerves of the LGA before and after pharmacologic antagonists; and (e) before and 30 min after the administration of pharmacologic antagonists. A Grass stimulator (Model S48; Grass Instruments, Quincy, Mass.) was used to stimulate the cervical vagus nerves and sympathetic nerves. This was accomplished by placing wire electrodes around the vagus nerve or the sympathetic nerves. Peripheral or central ends were stimulated separately. The stimulation parameters were 5-s trains of 10-V square-wave pulses, 0.5 ms in duration and 5-40 Hz in frequency (5, 10, 20, and 40 Hz). Pharmacologic studies were performed by using i.v. doses of the following pharmacologic agents: atropine sulfate, 30 kg/kg (Elli Lilly and Company, Indianapolis, Ind.); hexamethoniurn hydrochloride, 40 mg/kg (City Chemical Corp., New York, N.Y.); phentolamine, 1 mg/kg (Ciba Pharmaceutical Company, Sumit, N.J.); propranolol, 1 mgikg (Ayerst Laboratories, New York, N.Y.); nicotine sulfate, 100 pg/kg (Sigma Chemical Corporation, St. Louis, MO.); bethanechol, 40 p,g/kg (Merck, Sharp, & Dohme, West Point, Pa.); phenylephrine, 50 p.g/kg (Robinson Laboratories, San Francisco, Calif.); isoproterenol, 4 pg/kg (Winthrop Laboratories, New York, N.Y.); reserpine, 3 mg/kg intraperitoneally 48 and 24 h before the experiments (Ciba Geigy Company, Summit, N.J.); tyramine, 100 pg/kg (Aldrich Chemical Corporation, Metuchen, N.J.); TTX (Calbiochem, San Diego, Calif.) was given in small doses of 2-4 &kg
682
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40
,
k
1 min
I
bi
0 Figure 1. Tracing illustrates the LES contraction induced by electrical stimulation (10 Hz, 10 V) of both central ends of the cervical vagus nerve. The black dot indicates the timing and duration of the stimulus.
every 5-10 min, depending on the blood-pressure response. Successive doses of TTX were given until the LES response to vagal stimulation and esophageal distention with a small balloon were completely abolished. Each pharmacologic antagonist was administered intravenously over a 30-s period; LES pressures were measured 30 min later when arterial blood pressure was stable. At this time, the maximal dose of its respective agonist caused no changes in LES pressures. The LES response to electrical stimulation or pharmacologic agonists was determined at the peak or nadir of the pressure change within a lo-min period. It was expressed as absolute LES pressure or as the change in pressure (AP) in millimeters of mercury above or below control pressures obtained during a lo-min period preceeding the stimulation. Values are expressed as mean 2 SE. Paired and unpaired Student’s t-tests were applied to determine the significance of these differences with the two-tailed p-values.
Results Stimulation
of Extrinsic
Nerves
Electrical stimulation of both central ends of the cervical vagus nerves caused LES contraction in
19 out of 25 (76%) of the animals studied initially. Lower esophageal sphincter contraction started shortly after the beginning of the stimulation and continued 30 s to 1 min after cessation of the stimulus (Figure 1). Stimulation of the left or right vagus nerve in the presence of an intact contralateral nerve also resulted in LES contraction. Frequencyresponse studies showed that maximal LES response was obtained with 40 Hz and 10 V (Figure 2). The mean + SE of the LES contraction in these 19 animals is shown in Table 1. This LES response was blocked by section of the spinal cord above the brachial plexus (Table 2). Moreover, as shown in Table 3, this LES contraction was blocked by TTX (p < O.OOl), by a combination of hexamethonium and atropine (p < O.OOl), and by phentolamine (p < 0.001). It was not, however, antagonized by atropine alone (p < 1.0). The LES response, however, was abolished after catecholamine depletion with reserpine. Reserpine-treated animals were lethargic and hypotensive, with arterial blood pressures ranging from 110 to 90 mmHg systolic and from 50 to 40 mmHg diastolic, and developed diarrhea. The completeness of the catecholamine depletion was tested with tyramine. Tyramine causes LES contraction (12), which was not observed in these animals after treatment with reserpine.
Table
1.
Electrical
Stimulation
of Extrinsic
Nerves on the Lower Esophageal
Excitatory Sphincter
AP (mmHg)
n Cervical vagus Splanchnic nerves Periarterial sympathetics of LGA
FREQUENCY Figure 2. Frequency-response studies of electrical stimulation (5-40 Hz) of both central ends of the cervical vagus nerves. Values are mean 2 SE.
28
19
Peripheral end 34.3 2 4.0 24.5 k 8.0
n 19 22 19
Central end 11.8 k 1.1" 27.4 k 3.3 0
n = Number of experiments. “Values are mean ? SE of peak pressure increases (BP) above basal pressures during electrical stimulation. The parameters of electrical stimulation were IO Hz, 10 v.
NEURAL CONTROL OF THE CAT LES
April 1982
Table 2.
683
Effect of Extrinsic Denervation on the Lower Esophageal Sphincter Response to Stimulation of the Central Cervical Vagus Nerve
Ends
of the
AP (mmHg)b n Spinal cord Splanchnic nerves Periarterial sympathetics of LGA
6 6 6
Control
Nerve section
P’:
0.001 13.2 ‘- 1.0" 0 12.8? 1.6 12.8t 1.4 1 13.02 0.9 11.82 1.8 0.40 0
20
40
n = Number of experiments. ” Values are mean ? SE of the LES response to electrical stimulation (10 Hz, 10 V). b Indicates pressure changes with respect to basal pressures. ’ Paired Student’s t-test of the difference in the response before and after nerve section.
Figure 4. Frequency-response studies of the electrical stimulation (S-40 Hz) of the peripheral and central ends of the splanchnic nerves. Values are mean t SE.
Stimulation of the peripheral or central ends of the splanchnic nerves also caused LES contraction (Figure 3). This contraction occurred shortly after the onset and continued for
O.OOl), by phentolamine (Figure 6; < O.OOl), and by TTX at the dose of 12 kg/kg (Figure 7; p < 0.001). Furthermore, catecholamine depletion with reserpine abolished completely the LES response to stimulation (10 and 40 Hz) of either splanchnic end (p < 0.001). This neural stimulation was not blocked, however, by atropine alone at doses of 30 pg/kg (p < 0.70) or by propranolol (p < 0.90; Table 4). Because some neural branches arising from the left celiac ganglion run through the periadrenal connective tissue, the peripheral end of the splanchnic nerves was stimulated electrically before and after bilateral adrenalectomy. Lower esophageal sphincter
FREQUENCY
200-
50f aG (n= 2
01 :
200 150 100
Figure
3. Tracing illustrates the LES contraction in response to electrical stimulation (10 Hz, 10 V) of the peripheral end (middle tracing) and central end (lower tracing) of the splanchnic nerves. Arterial blood pressure changes are shown in the upper tracing. Arrow and black dot indicates timing and duration of the stimulus.
684
BEHAR
ET AL.
GASTROENTEROLOGY
PERIPHERAL
IOO-
END
T
clBASAL I 10 Hz
60 --
I
^o ; 60--
only with TTX (12 puglkg) and phentolamine (p < 0.001, Table 5). It was not antagonized by hexamethonium and atropine (p < 0.80). Electrical stimulation of the central end of the LGA did not affect the LES pressures.
40 Hz Genesis of Basal Lower Esophageal Sphincter Pressures
E $) w J
Extrinsic denervation did not affect basal LES pressures (Table 6). No change in LES pressure was observed after bilateral section of the cervical or
40--
PERIPHERAL
t
100 CENTRAL 24C
Vol. 82, No. 4
T
END
IT
END
j-l BASAL_
60 G I
2oc I--
l-.
t
60
:
40
I--
W J I--
2c I--
0 I-
CENTRAL END
0 CONTROL
HEXAME+THONIUM ATROPINE
Figure
5. Lower esophageal sphincter response to electrical stimulation of the peripheral and central ends of the splanchnic nerves before and after the administration of hexamethonium and atropine (p < 0.001). Values are mean f SE of absolute LES pressures.
60
pressures were not different before and after adrenalectomy (p < 0.40, Table 4). Stimulation of the peripheral end of the periarterial sympathetics of the LGA caused contraction of the LES (Figure 8). This contraction was similar to that observed after stimulation of the central end of the vagus nerve and of either the peripheral or central end of the splanchnic nerves. It occurred shortly after the onset of the stimulus and continued for
40
20
0 CONTROL
Figure
PHI lNTOLAMlNE . _._ tlmg/Kg)
sphincter contraction induced by 6. Lower esophageal electrical stimulation of the peripheral and central ends of the splanchnic nerves before and after phentolamine (p < 0.001). Values are mean rt SE of absolute LES pressures.
April 1982
NEURAL
PERIPHERAL
END
0 BASAL
CONTROL
685
OF THE CAT LES
affect basal LES pressures. There was a brief reduction in pressures after the administration of hexamethonium that correlated well with the fall in arterial blood pressure. Lower esophageal sphincter pressures, however, recovered 0.5 h after the administration of this antagonist despite the persistent arterial hypotension; furthermore, the LES pressure response to nicotine was still inhibited.
Discussion
CENTRAL
This study presents evidence for the existence of an excitatory reflex arc with an afferent limb in the vagal pathway and an eff erent limb in the sympathetic pathway. Several findings support this conclusion. First, the timing and duration of the LES contraction in response to electrical stimulation of the central ends of the cervical vagus and of the splanchnic nerves were similar. The LES response occurred during electrical stimulation and lasted for
END
120-
-
P
60--
E - 60-p % 3
40--
20--
o-
CONTROL
TTX
(12JbOMOI Figure
7. Lower esophageal sphincter response to electrical stimulation of the peripheral and central ends of the splanchnic nerves before and after the administration of TTX (p < 0.001). Values are mean 2 SE of absolute LES pressures.
thoracic vagus nerve (p < 0.50). Similarly, basal LES pressures were not influenced by section of the splanchnic nerves or celiac ganglion (p < MO), or the periarterial sympathetics of the LGA (p < O.BO), or of the cervical spinal cord (p < 0.60). Pharmacologic analysis of basal LES pressures showed that pressures decreased after TTX at doses of 12 &kg (p < 0.001)or after phentolamine administration (p < 0.001; Table 7). Increasing the dose of TTX from 12 to 36 pgkg, which effectively abolished LES relaxation in response to esophageal distention, did not reduce LES pressures any further. Atropine alone (p < 0.10) or in combination with hexamethonium (p < 0.10) or propranolol (p < 0.30) did not
Table
3.
Effect of Pharmacologic Antagonists on the Lower Esophageal Sphincter Response to Electrical Stimulation of Both Central Ends of the Cervical Vagus Nerve
Control
7
{ 10.1 ;
I
6
l,5.0:2.2
Reserpine
5
Phentolamine
1.71
()ool
I
Control TTX
0.001
I 0
-
n = Number of experiments. o Values sre mean f SE of pressure changes (A) above basal pressures in response to electrical stimulation (10 Hz, 10 V). b Paired Student’s t-test applied to the difference in the LES response before and after pharmacologic antagonists.
686
GASTROENTEROLOGY
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Table
4.
Effect of Pharmacologic Antagonists Contraction Induced by Splanchnic
and Bilateral Adrenalectomy Nerve Stimulation
on the Lower Esophageal
Vol. 82. No. 4
Sphincter
AP ImmHgl Peripheral n 5
5
Control
lo Hz I
40 Hz
I
110 + 26
48.0 2 13.2
107 k 26
46.5 f 11.3
64.2 + 8.9
51.7 -c 10.3
67.0 + 11.4
0.70
I
39.8
I 25.5 k 3.2
37.7 + 5.8 j
0.40
lo Hz --.-~-__--. 46.4 k 15
83.0 5 20
54.0 ? 17.4
85.6 + 23
26.8 -t 2.9
43.2 t 5.6
27.2 t 3.3
40.2 2 2.8
/
-
I
-
’ Values are mean f SE. b Paired Student’s t-test was used to determine before and after pharmacologic or surgical treatment.
nerves. These findings also suggest the possibility of a second reflex arc present in the sympathetic pathway, because stimulation of the central end of the left splanchnic nerves caused LES contraction. Further, in agreement with anatomic studies (13), our results indicate that the LES receives sympathetic fibers from nerves following gastric arteries from the celiac ganglion. The LES contraction induced by stimulation of this reflex arc was antagonized when the animals were treated with a combination of atropine and hexamethonium or with phentolamine. These findings reveal the presence of preganglionic cholinergic fibers (14) and postganglionic adrenergic neurons that stimulate a-receptors at the LES circular muscle. Preganglionic fibers seem to synapse with adrenergic neurons at the celiac ganglion because their stimulation was antagonized by the ganglionic blocker, in combination with atropine. hexamethonium, These observations are consistent with previous studies on the sympathetic innervation of the gastrointestinal tract (15,16) that have shown that stimulation of the sympathetic nerves, with few exceptions, excites sphincteric and inhibits nonsphincteric structures. They are also supported by previous histologic studies showing that noradrenergic termi-
Figure
k 1.9
0.70
[ 28.2 t 6.8
n = Number of experiments.
the LES response
P”
53.0 + 13.0"
15
Bilateral adrenalectomy
Central end -___---_-
end
8. Tracing
illustrates the LES response to electrical stimulation (10 Hz, 10 V) of the peripheral end of the left gastric artery.
Black dot indicates the timing tion of the stimulus.
and dura-
the significance
C n
20-
Pb I
0.50
0.90
of the differences
in
nals are found in the circular muscle of the sphincter and that noradrenergic neurons are present outside the gastrointestinal tract (13). Stimulation of the splanchnic nerves contracts the LES due to direct action of norepinephrine released from postganglionic axon terminals on the circular smooth muscle (17). The action of the endogenously released norepinephrine during stimulation of the splanchnic nerves was in agreement with that of exogenous norepinephrine. In the cat, exogenous norepinephrine caused LES contraction by acting directly on the circular muscle because TTX does not prevent this action (12). The LES response during excitation of this reflex arc is not mediated by stimulation of the adrenal glands. This possibility was entertained because glucagon caused LES contraction by releasing epinephrine or norepinephrine from the adrenal glands (12). Bilateral adrenalectomy blocked the effect of glucagon. In spite of the anatomic proximity of the sympathetic branches arising from the celiac ganglia to the adrenal glands, bilateral adrenalectomy did not impair the LES response to sympathetic stimulation. Basal LES tone is not dependent on the integrity of this reflex arc. Basal LES pressures remain un-
w 60-
II v) o!!i
---
40 Hz
60 SECONDS
NEURAL CONTROL OF THE CAT LES
April 1982
Table
6.
Effect of Section of Extrinsic Lower Esophageal Sphincter
‘K--v-l0
Cervical vagus nerves Thoracic vagus nerves Spinal cord Splanchnic nerves Periarterial sympathetics of the LGA
40
20
FREQUENCY
Figure 9. Frequency-response studies of electrical stimulation (5-40 Hz) of the peripheral end of the left gastric artery. Values are mean ? SE.
changed after interruption of this excitatory reflex at the cervical vagus nerves or at the spinal cord. These observations are supported by the finding that hexamethonium and atropine, which effectively eliminate the contribution of preganglionic fibers of this reflex, do not affect basal LES pressures. It is still possible, however, that the postganglionic adrenergic neurons of the sympathetic pathway may contribute to the genesis of LES tone. Complete LES sympathectomy was not achieved by section of the left splanchnic nerves or of the periarterial sympathetics of the LGA. The finding that phentolamine, an cY-adrenergic antagonist, caused a moderate but consistent reduction of basal LES pressures supports this assumption. It was further confirmed by the studies with TTX. These phentolamine or TTX effects are not related to a concomitant fall in arterial blood pressure. A similar blood pressure fall due to arterial bleeding produced only a transient reduction
Nerves on Basal Pressures
Control lmmHrrl
n 9 3 6 7 7
30.8 36.4 34.0 48.3 52.4
687
Nerve section
-+ 4.8’ % 3.8 C 6.4 + 3.9 ‘- 4.7
37.2 37.9 30.2 45.6 51.3
t c t 2 ?
Pb
4.2 0.20 4.7 0.50 5.1 0.60 2.4 0.40 4.7 0.80
n = Number of experiments. ’ Values are mean ? SE of absolute LES pressures. b Paired Student’s t-test comparing pressures before and 30 min after nerve section.
in LES pressures (18). Alternative explanations, however, are possible. As in the opossum, the LES circular muscle may be primarily responsible for its tone, but it is regulated by a balance of excitatory and inhibitory neural influences. It is conceivable that phentolamine antagonizes the excitatory nerves enhancing the influence of TTX resistent inhibitory nerves (19). These studies do not support the existence of a postganglionic cholinergic neuron within this reflex arc as effective doses of atropine alone did not
Table
7.
Effect of Pharmacologic Antagonists on Basal Lower Esophageal Sphincter Pressures
Control
n
Basal pressures (nunHal
% Change
)
6
( “,;:r 1 ::;)
-28.3”
)
7
( ::::
1
9
(;;:;:I;)
1
9
( :;I’, : :I:
)
-12.2
]
8
[ :I:;;:;;
)
--25.8
- -- Pb 0.001
Tetrodotoxin (12 pg/kg)
Table
-_..
5.
_.
Effect of Pharmacologic Antagonists on the Lower Esophageal Sphincter Response to Electrical Stimulation of Periarteriaf Sympathetics of Left Gastric Artery --LGA stimulation n
__. _.-_.._IControl
_-_- __.~__..
AP (mmHg) - ._-_-
Atropine (30 wg/kg) Control
6
6
Hexamethonium and atropine
I ““‘”
I
0.001
6.6
TTX (12 pg/kg) Control
0.001 7
( 1°.Y
)
--8.3
f5.5
1.7 )
I
n = Number of experiments. a Mean 2 SE of LES pressure changes above basal pressures. b Paired Student’s t-test was applied to the LES response to LGA stimulation (10 Hz, 10 V) before and 30 min after the administration of each pharmacologic antagonist.
0.10
0.50
Hexamethonium
Control
Control
: ‘:I;
Control
Hexamethonium and atropine
Phentolamine (1 mg/kgl
Control
0.40
0.001
Phentolamine (1 mglkg) Control
0.30
Propranolol (1 mg/kgl
n = Number of experiments. a Numbers are mean 2 SE of absolute LES pressures. b Paired Student’s t-test comparing pressures before and after the administration of pharmacologic antagonists. c Values represent the percent of pressure change induced by drugs; control pressures are counted as 100%.
668
GASTROENTEROLOGY Vol.
BEHAR ET AL.
antagonize the LES contraction in response to vagal or sympathetic stimulation even though they effectively blocked the maximal dose of exogenous bethanechol. Only after the addition of the ganglionic blocker, hexamethonium, did the importance of the cholinergic receptors become apparent. These receptors appeared to be present at the celiac ganglion cells, as the stimulation of the postganglionic periarterial fibers of the LGA was not affected by these antagonists. These studies do not support the hypothesis that a postganglionic cholinergic neuron participates in the genesis of basal LES tone. There is, however, indirect evidence of the existence of such a neuron in the cat because the LES contraction induced by 5-hydroxytryptamine is partially blocked by atropine (Behar J: unpublished observation). Other in vitro (20) and in vivo (21,X?) studies on the cat LES have suggested the presence of postganglionic cholinergic neurons contributing to the genesis of sphincter tone. Different methodology and experimental design may explain these discrepancies. The anchored manometric probe used in this study allowed us to obtain continuous and accurate LES measurements throughout the experiment. We have observed transient reductions in LES pressures 5 or 10 min after the administration of atropine; LES pressures, however, returned to baseline levels while there still was an effective blockade of a maximal dose of bethanechol. Lower esophageal sphincter relaxation was never observed during stimulation of this reflex arc even after blocking excitatory receptors. Sphincter inhibition during stimulation of the central end of the splanchnic nerves has been recently shown in the cat (22). This study differed from ours in at least two significant aspects: [a) LES pressures measured were only lo-15 mmHg, whereas in our laboratory we found pressures of 44 ? 12 mmHg (mean ? SE in 412 animals). Other studies in the same animal species have shown results similar to ours (23). This pressure difference cannot be easily explained because it cannot be accounted for by either flow rate or by diameter of the catheter. (b) The manometric catheter was anchored at the LES by placing a small arterial clip, which in our preliminary experiments was not sufficient to anchor the pressure sensor firmly within the LES and prevent movements of the catheter. In conclusion, the present studies show the existence of an excitatory reflex arc with an afferent vagal and an efferent sympathetic pathway to the cat LES. It consists of preganglionic cholinergic fibers and postganglionic adrenergic neurons that stimulate a-receptors. Although its physiologic role remains unclear, it is possible that the postganglionic adrenergic neurons may contribute to the genesis of
basal LES tone or to its regulation tonic neural inhibitory influences.
82, No. 4
in balance
with
References Winan CS, Harris LD. Quantitation of lower esophageal sphincter competence. Gastroenterology 1967;52:773-8. 2. Biancani P, Zabinski MP, Behar J. Pressure, tension, and force of closure on the human lower esophageal sphincter and esophagus. J Chn Invest 1975;56:476-83. 3. Christensen J, Freeman BW, Miller JK. Some physiological characteristics of the esophagogastric junction in the opossum. Gastroenterology 1973;64:1119-25, 4. Biancani P, Kerstein M, Weiss M, et al. Force-length relationships of the lower esophageal sphincter (LES). (in press]. 5. Goyal RK, Rattan S. Genesis of basal sphincter pressure: effect of tetrodotoxin on lower esophageal sphincter pressure in opossum in vivo. Gastroenterology 1976;71:62-7. 6. Behar J, Kerstein M, Biancani P. Neural control of lower esophageal sphincter closure (abstr). Gastroenterology 1977; 72~1029. 7. Behar J, Kastendieck J. Studies on sphincter competence (abstr). Gastroenterology 1974;66:834. 8. Dodds WJ, Dent J, Hogan WJ, et al. The effect of atropine on esophageal motor function in man. Am J Physiol 1982;(in press). 9. Meissner AJ, Bowes KL, Zwick R, et al. Effect of motilin on the lower esophageal sphincter. Gut 1976;17:925-32. 10. Rattan S, Goyal RK. Neural control of the lower esophageal sphincter: influence of the vagus nerves. J Clin Invest 1974;54:899-906. 11. Behar J, Biancani P. Effect of cholecystokinin-octapeptide on lower esophageal sphincter. Gastroenterology 1977;73:57-61. 12. Behar J, Field S, Marin C. Effect of glucagon, secretin, and vasoactive intestinal polypeptide on the feline lower esophageal sphincter: mechanism of action. Gastroenterology 1979;77:1001-7. 13. Mitchell GAG. Anatomy of the autonomic nervous system. Edinburgh, London: Livstone, 1973. 14. Goyal RK, Rattan S. Nature of the vagal inhibitory innervation of the lower esophageal sphincter. J Clin Invest 1975; 55:1119-26. 15. Furness JB, Costa M. The adrenergic innervation of the gastrointestinal tract. Ergeb Physiol Biol Chem Exp Pharmakol 1979;69:1-51. 16. Gershon MD, Erde SM. The nervous system of the gut. Gastroenterology 1981;80:1571-94. 17. Botna GSM. The gastroesophageal junction. London: Churchiff, 1962. 18 LiCalzi LK, Biancani P, Behar J, et al. Effect of hemorrhagic hypotension and hypoventilation on lower esophageal sphincter pressure. Ann Surg 1980;192:53-7. 19. Carter RH. Resistance of tetrodotoxin in toad sympathetic nerves. J Pharm Pharmacol 1969;21:394-5. 20. Gonella J, Niel JP, Roman C. Vagal control of lower oesophageal sphincter motility in the cat. J Physiol (Lond) 1977; 273:647-64. 21. Eastwood GL, Beck BD, Caste11 DO, et al. Beneficial effect of indomethacin on acid-induced esophagitis in cats. Dig Dis Sci 1981;26:601-8. 22. Fournet J, Snape WJ, Cohen S. Sympathetic control of lower esophageal sphincter function in the cat. J Clin Invest 1979;63:562-70. 23. Eastwood GL, Caste11 DO, Higgs RH. Experimental esophagitis in cats impairs lower esophageal sphincter pressure. Gastroenterology 1975;69:146-53. 1.
1982;82:680-8
Neural Control of the Lower Esophageal Sphincter in the Cat: Studies on the Excitatory Pathways to the Lower Esophageal Sphincter JOSE BEHAR, MORRIS KERSTEIN, and PIER0 BIANCANI The Department of Medicine, Rhode Island Hospital and Brown University, Providence, Rhode Island, and the Department of Surgery, Tulane University, New Orleans, Louisiana
Lower esophageal sphincter pressures were measured with a 6-lumen catheter anchored at the high pressure point by a metallic pin. Electrical stimulation (5-40 Hz) of both central ends of the cervical vagus caused contraction of the lower eSOphQgeQ1 sphincter, which was antagonized by section of the spinal cord above the brachial plexus, by tetrodotoxin, by a combination of hexamethonium and atropine, by phentolamine, and by catecholamine depletion with reserpine. Stimulation (5-40 Hz] of peripheral or central ends of the left sphmchnic nerves also caused contraction of the lower esophageal sphincter. This sphincter response was antagonized by tetradotoxin, a combination of hexamethonium and atropine, phentolamine, and catecholamine depletion With reserpine. It was not affected by atropine alone or by propranolol. Lower esophageal sphincter relaxation was not observed even after the contraction was blocked with phQrmQcologic antagonists. Stimulation (5-40 Hz) of the peripheral end of the periarterial nerves of the left gastric artery also caused contraction of the sphincter and was antagonized only by tetrodotoxin and phentolamine. Atropine and hexamethonium did not affect this sphincter response. Extrinsic denervation of the lower esophageal sphincter by sectioning of the cervical vagus nerves, the spinal cord, the left sphmchnic nerves, or the left gastric artery did not affect basal sphincter pressures. BQSQ~ sphincter pressure was not affected by atropine alone, by Q combination of hexamethonium and atropine, or by Received June 29, 1981. Accepted November 12, 1981. Address requests for reprints to: Dr. Jose Behar, APC Building, Room 421, Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02902. The authors thank Carol Marin, Susan Field, and Judy Johnson for the laboratory assistance, and Lorie Klemer for her secretarial help. 0 1982 by the American Gastroenterological 0016-5085/82/040680-09$02.50
Association
propranolol. There was, however, a 25%-28% reduction with tetrodotoxin and phentolamine. These findings suggest the existence of an excitatory reflex arc with an afferent vagaJ and efferent sympathetic pathway. It consists of pregcmgbonic cholinergic fibers and postganglionic adrenergic neurons synapsing with Q-receptors at the muscle fiber. These adrenergic neurons may contribute to the genesis of bQSQ1 lower esophageal sphincter tone, or to its regulation in balance with tonic neural inhibitory influences. Lower esophageal sphincter (LES) closure is determined by tension of the circular muscle (1,2). The mechanism by which sphincter closure is maintained, however, has not been elucidated. In vitro studies using circular muscle strips from the LES of the opossum and cat have shown that at the gastroesophageal junction there is a specialized circular muscle layer with steeper length-tension curves than those from adjacent areas (3,4). In the opossum, in vivo basal sphincter pressures are only slightly or not at all affected by any of the neural antagonists, including tetrodotoxin (TTX) (5). Other in vivo studies (7-g), however, have presented indirect evidence that neural mechanisms may contribute to the genesis of sphincter tone. The magnitude of the neural contribution is different in various animal species. In the opossum, LES innervation does not appear to play any role in the genesis of basal tone (5); while in the cat, we have shown a reduction of 25%-28% in LES pressures after the administration of TTX or of an a-adrenergic antagonist such as phentolamine (6). In conscious humans, atropine and probanthine decrease basal sphincter pressures significantly (7,B). Similarly, the dog LES is affected by atropine (9). Excitatory pathways to the LES have been shown in both the opossum and the cat by using electrical stimulation of extrinsic nerves (6,10,11). Stimulation
April
1982
of afferent vagal fibers and of periarterial sympathetic fibers of the left gastric artery (LGA) caused LES contraction. The purposes of the present study were: to investigate the excitatory neural pathways to the cat LES, to determine the nature of the neural fibers present, and to assess their contribution to basal sphincter tone.
Materials and Methods These studies were performed in 86 adult cats of either sex weighing 3-4 kg. After fasting for 16 h, the animals were anesthetized intramuscularly with ketamine hydrochloride (30 mg/kg body wt) and were kept lightly anesthetized with maintenance doses of 5-10 mgikg throughout the experiment. Controlled ventilation was maintained in all animals with a respirator (Harvard Apparatus Co., South Natick, Mass.) via a tracheostomy tube. All animals were supine when studied. Normal saline with 5% dextrose was slowly infused through an iv. cannula. Arterial blood pressure was recorded by cannulating the femoral artery with a catheter connected to a Statham pressure transducer. Esophageal, LES, and gastric pressures were recorded with a low-compliance system using a constant hydraulic infusion pump. A water-filled, g-lumen catheter assembly was perfused at the rate of 0.3 mllmin, connected to a Statham pressure transducer and a 12-channel Grass polygraph. The rate of pressure increase of this system with occlusion of the manometric probe (>lOO mmHg) was greater than the observed changes in LES pressure. The catheter assembly consisted of six polyvinyl tubes (OD of each = 1.6 mm), glued on the anterior and posterior aspects of a hollow polyvinyl tube (OD = 3.4 mm). The dimensions of the entire assembly were 6.6 mm in its longest and 3.4 mm in its shortest diameter. The catheter was introduced into the stomach through the mouth. The LES was located with two gradual pullthroughs by using the four side openings, 1 mm apart. Gastric pressure was measured by the side opening 1 cm distal, and esophageal pressure was measured with the side opening 2 cm proximal to the four central openings. After an abdominal midline incision, the gastroesophageal junction was exposed, and the probe was anchored at this level by inserting a thin, metallic pin through the LES wall and lateral side of the hollow tubing. The pin was covered with small corks at both ends to avoid damage to the adjacent organs. The pin-anchored catheter assembly permitted evaluation of the LES pressures at the same level and radial orientation throughout the experiment. Lower esophageal sphincter pressure throughout the experiment was stable. After the abdominal incision was covered with wet gauze, basal sphincter pressure was allowed to stabilize for 30 min before beginning the study. The LES was defined as a zone of high pressure that relaxed during electrical stimulation of the peripheral end of the vagus nerve or during distention of a small rubber balloon placed in midesophagus. Placement of the metallic pin through the LES did not affect LES function (11).Water infused
NEURAL CONTROL OF THE CAT LES
681
into the stomach through the six side openings was allowed to drain by gravity through the hollow core tube. This continuous drainage prevented distention of the stomach.
Experimental
Studies
The vagus nerves were identified and carefully exposed at the level of the neck in all studies. For those studies involving the splanchnic nerves, the left celiac ganglia and its branches were identified and carefully dissected at the origin of the celiac artery. The left splanchnit nerves were secured by tying two silk sutures, and they were then sectioned into peripheral ends going into the ganglia and central ends leading back to the sympathetic chain and spinal cord. The spinal cord was severed above the level of the brachial plexus for those studies involving spinal cord section. The completeness of the section was determined by finding that reflexes in all four extremities were abolished. For studies involving the LGA, the artery was identified and isolated with its periarterial connective tissue and plexus. It was secured by tying two silk sutures around it, and then severed into a peripheral end leading to the gastroesophageal junction and a central end to the porta hepatis. Lower esophageal sphincter pressures were monitored under the following conditions: [a) electrical stimulation of both central ends of the cervical vagus nerve before and after pharmacologic antagonists; (b) electrical stimulation of central and peripheral ends of both left greater and lesser splanchnic nerves, before and after treatment with pharmacologic antagonists; (c) before and after spinal cord section in basal conditions and during electrical stimulation; (d) electrical stimulation of the periarterial sympathetic nerves of the LGA before and after pharmacologic antagonists; and (e) before and 30 min after the administration of pharmacologic antagonists. A Grass stimulator (Model S48; Grass Instruments, Quincy, Mass.) was used to stimulate the cervical vagus nerves and sympathetic nerves. This was accomplished by placing wire electrodes around the vagus nerve or the sympathetic nerves. Peripheral or central ends were stimulated separately. The stimulation parameters were 5-s trains of 10-V square-wave pulses, 0.5 ms in duration and 5-40 Hz in frequency (5, 10, 20, and 40 Hz). Pharmacologic studies were performed by using i.v. doses of the following pharmacologic agents: atropine sulfate, 30 kg/kg (Elli Lilly and Company, Indianapolis, Ind.); hexamethoniurn hydrochloride, 40 mg/kg (City Chemical Corp., New York, N.Y.); phentolamine, 1 mg/kg (Ciba Pharmaceutical Company, Sumit, N.J.); propranolol, 1 mgikg (Ayerst Laboratories, New York, N.Y.); nicotine sulfate, 100 pg/kg (Sigma Chemical Corporation, St. Louis, MO.); bethanechol, 40 p,g/kg (Merck, Sharp, & Dohme, West Point, Pa.); phenylephrine, 50 p.g/kg (Robinson Laboratories, San Francisco, Calif.); isoproterenol, 4 pg/kg (Winthrop Laboratories, New York, N.Y.); reserpine, 3 mg/kg intraperitoneally 48 and 24 h before the experiments (Ciba Geigy Company, Summit, N.J.); tyramine, 100 pg/kg (Aldrich Chemical Corporation, Metuchen, N.J.); TTX (Calbiochem, San Diego, Calif.) was given in small doses of 2-4 &kg
682
BEHAR ET AL.
GASTROENTEROLOGY Vol. 82, No. 4
40
,
k
1 min
I
bi
0 Figure 1. Tracing illustrates the LES contraction induced by electrical stimulation (10 Hz, 10 V) of both central ends of the cervical vagus nerve. The black dot indicates the timing and duration of the stimulus.
every 5-10 min, depending on the blood-pressure response. Successive doses of TTX were given until the LES response to vagal stimulation and esophageal distention with a small balloon were completely abolished. Each pharmacologic antagonist was administered intravenously over a 30-s period; LES pressures were measured 30 min later when arterial blood pressure was stable. At this time, the maximal dose of its respective agonist caused no changes in LES pressures. The LES response to electrical stimulation or pharmacologic agonists was determined at the peak or nadir of the pressure change within a lo-min period. It was expressed as absolute LES pressure or as the change in pressure (AP) in millimeters of mercury above or below control pressures obtained during a lo-min period preceeding the stimulation. Values are expressed as mean 2 SE. Paired and unpaired Student’s t-tests were applied to determine the significance of these differences with the two-tailed p-values.
Results Stimulation
of Extrinsic
Nerves
Electrical stimulation of both central ends of the cervical vagus nerves caused LES contraction in
19 out of 25 (76%) of the animals studied initially. Lower esophageal sphincter contraction started shortly after the beginning of the stimulation and continued 30 s to 1 min after cessation of the stimulus (Figure 1). Stimulation of the left or right vagus nerve in the presence of an intact contralateral nerve also resulted in LES contraction. Frequencyresponse studies showed that maximal LES response was obtained with 40 Hz and 10 V (Figure 2). The mean + SE of the LES contraction in these 19 animals is shown in Table 1. This LES response was blocked by section of the spinal cord above the brachial plexus (Table 2). Moreover, as shown in Table 3, this LES contraction was blocked by TTX (p < O.OOl), by a combination of hexamethonium and atropine (p < O.OOl), and by phentolamine (p < 0.001). It was not, however, antagonized by atropine alone (p < 1.0). The LES response, however, was abolished after catecholamine depletion with reserpine. Reserpine-treated animals were lethargic and hypotensive, with arterial blood pressures ranging from 110 to 90 mmHg systolic and from 50 to 40 mmHg diastolic, and developed diarrhea. The completeness of the catecholamine depletion was tested with tyramine. Tyramine causes LES contraction (12), which was not observed in these animals after treatment with reserpine.
Table
1.
Electrical
Stimulation
of Extrinsic
Nerves on the Lower Esophageal
Excitatory Sphincter
AP (mmHg)
n Cervical vagus Splanchnic nerves Periarterial sympathetics of LGA
FREQUENCY Figure 2. Frequency-response studies of electrical stimulation (5-40 Hz) of both central ends of the cervical vagus nerves. Values are mean 2 SE.
28
19
Peripheral end 34.3 2 4.0 24.5 k 8.0
n 19 22 19
Central end 11.8 k 1.1" 27.4 k 3.3 0
n = Number of experiments. “Values are mean ? SE of peak pressure increases (BP) above basal pressures during electrical stimulation. The parameters of electrical stimulation were IO Hz, 10 v.
NEURAL CONTROL OF THE CAT LES
April 1982
Table 2.
683
Effect of Extrinsic Denervation on the Lower Esophageal Sphincter Response to Stimulation of the Central Cervical Vagus Nerve
Ends
of the
AP (mmHg)b n Spinal cord Splanchnic nerves Periarterial sympathetics of LGA
6 6 6
Control
Nerve section
P’:
0.001 13.2 ‘- 1.0" 0 12.8? 1.6 12.8t 1.4 1 13.02 0.9 11.82 1.8 0.40 0
20
40
n = Number of experiments. ” Values are mean ? SE of the LES response to electrical stimulation (10 Hz, 10 V). b Indicates pressure changes with respect to basal pressures. ’ Paired Student’s t-test of the difference in the response before and after nerve section.
Figure 4. Frequency-response studies of the electrical stimulation (S-40 Hz) of the peripheral and central ends of the splanchnic nerves. Values are mean t SE.
Stimulation of the peripheral or central ends of the splanchnic nerves also caused LES contraction (Figure 3). This contraction occurred shortly after the onset and continued for
O.OOl), by phentolamine (Figure 6; < O.OOl), and by TTX at the dose of 12 kg/kg (Figure 7; p < 0.001). Furthermore, catecholamine depletion with reserpine abolished completely the LES response to stimulation (10 and 40 Hz) of either splanchnic end (p < 0.001). This neural stimulation was not blocked, however, by atropine alone at doses of 30 pg/kg (p < 0.70) or by propranolol (p < 0.90; Table 4). Because some neural branches arising from the left celiac ganglion run through the periadrenal connective tissue, the peripheral end of the splanchnic nerves was stimulated electrically before and after bilateral adrenalectomy. Lower esophageal sphincter
FREQUENCY
200-
50f aG (n= 2
01 :
200 150 100
Figure
3. Tracing illustrates the LES contraction in response to electrical stimulation (10 Hz, 10 V) of the peripheral end (middle tracing) and central end (lower tracing) of the splanchnic nerves. Arterial blood pressure changes are shown in the upper tracing. Arrow and black dot indicates timing and duration of the stimulus.
684
BEHAR
ET AL.
GASTROENTEROLOGY
PERIPHERAL
IOO-
END
T
clBASAL I 10 Hz
60 --
I
^o ; 60--
only with TTX (12 puglkg) and phentolamine (p < 0.001, Table 5). It was not antagonized by hexamethonium and atropine (p < 0.80). Electrical stimulation of the central end of the LGA did not affect the LES pressures.
40 Hz Genesis of Basal Lower Esophageal Sphincter Pressures
E $) w J
Extrinsic denervation did not affect basal LES pressures (Table 6). No change in LES pressure was observed after bilateral section of the cervical or
40--
PERIPHERAL
t
100 CENTRAL 24C
Vol. 82, No. 4
T
END
IT
END
j-l BASAL_
60 G I
2oc I--
l-.
t
60
:
40
I--
W J I--
2c I--
0 I-
CENTRAL END
0 CONTROL
HEXAME+THONIUM ATROPINE
Figure
5. Lower esophageal sphincter response to electrical stimulation of the peripheral and central ends of the splanchnic nerves before and after the administration of hexamethonium and atropine (p < 0.001). Values are mean f SE of absolute LES pressures.
60
pressures were not different before and after adrenalectomy (p < 0.40, Table 4). Stimulation of the peripheral end of the periarterial sympathetics of the LGA caused contraction of the LES (Figure 8). This contraction was similar to that observed after stimulation of the central end of the vagus nerve and of either the peripheral or central end of the splanchnic nerves. It occurred shortly after the onset of the stimulus and continued for
40
20
0 CONTROL
Figure
PHI lNTOLAMlNE . _._ tlmg/Kg)
sphincter contraction induced by 6. Lower esophageal electrical stimulation of the peripheral and central ends of the splanchnic nerves before and after phentolamine (p < 0.001). Values are mean rt SE of absolute LES pressures.
April 1982
NEURAL
PERIPHERAL
END
0 BASAL
CONTROL
685
OF THE CAT LES
affect basal LES pressures. There was a brief reduction in pressures after the administration of hexamethonium that correlated well with the fall in arterial blood pressure. Lower esophageal sphincter pressures, however, recovered 0.5 h after the administration of this antagonist despite the persistent arterial hypotension; furthermore, the LES pressure response to nicotine was still inhibited.
Discussion
CENTRAL
This study presents evidence for the existence of an excitatory reflex arc with an afferent limb in the vagal pathway and an eff erent limb in the sympathetic pathway. Several findings support this conclusion. First, the timing and duration of the LES contraction in response to electrical stimulation of the central ends of the cervical vagus and of the splanchnic nerves were similar. The LES response occurred during electrical stimulation and lasted for
END
120-
-
P
60--
E - 60-p % 3
40--
20--
o-
CONTROL
TTX
(12JbOMOI Figure
7. Lower esophageal sphincter response to electrical stimulation of the peripheral and central ends of the splanchnic nerves before and after the administration of TTX (p < 0.001). Values are mean 2 SE of absolute LES pressures.
thoracic vagus nerve (p < 0.50). Similarly, basal LES pressures were not influenced by section of the splanchnic nerves or celiac ganglion (p < MO), or the periarterial sympathetics of the LGA (p < O.BO), or of the cervical spinal cord (p < 0.60). Pharmacologic analysis of basal LES pressures showed that pressures decreased after TTX at doses of 12 &kg (p < 0.001)or after phentolamine administration (p < 0.001; Table 7). Increasing the dose of TTX from 12 to 36 pgkg, which effectively abolished LES relaxation in response to esophageal distention, did not reduce LES pressures any further. Atropine alone (p < 0.10) or in combination with hexamethonium (p < 0.10) or propranolol (p < 0.30) did not
Table
3.
Effect of Pharmacologic Antagonists on the Lower Esophageal Sphincter Response to Electrical Stimulation of Both Central Ends of the Cervical Vagus Nerve
Control
7
{ 10.1 ;
I
6
l,5.0:2.2
Reserpine
5
Phentolamine
1.71
()ool
I
Control TTX
0.001
I 0
-
n = Number of experiments. o Values sre mean f SE of pressure changes (A) above basal pressures in response to electrical stimulation (10 Hz, 10 V). b Paired Student’s t-test applied to the difference in the LES response before and after pharmacologic antagonists.
686
GASTROENTEROLOGY
BEHAR ET AL.
Table
4.
Effect of Pharmacologic Antagonists Contraction Induced by Splanchnic
and Bilateral Adrenalectomy Nerve Stimulation
on the Lower Esophageal
Vol. 82. No. 4
Sphincter
AP ImmHgl Peripheral n 5
5
Control
lo Hz I
40 Hz
I
110 + 26
48.0 2 13.2
107 k 26
46.5 f 11.3
64.2 + 8.9
51.7 -c 10.3
67.0 + 11.4
0.70
I
39.8
I 25.5 k 3.2
37.7 + 5.8 j
0.40
lo Hz --.-~-__--. 46.4 k 15
83.0 5 20
54.0 ? 17.4
85.6 + 23
26.8 -t 2.9
43.2 t 5.6
27.2 t 3.3
40.2 2 2.8
/
-
I
-
’ Values are mean f SE. b Paired Student’s t-test was used to determine before and after pharmacologic or surgical treatment.
nerves. These findings also suggest the possibility of a second reflex arc present in the sympathetic pathway, because stimulation of the central end of the left splanchnic nerves caused LES contraction. Further, in agreement with anatomic studies (13), our results indicate that the LES receives sympathetic fibers from nerves following gastric arteries from the celiac ganglion. The LES contraction induced by stimulation of this reflex arc was antagonized when the animals were treated with a combination of atropine and hexamethonium or with phentolamine. These findings reveal the presence of preganglionic cholinergic fibers (14) and postganglionic adrenergic neurons that stimulate a-receptors at the LES circular muscle. Preganglionic fibers seem to synapse with adrenergic neurons at the celiac ganglion because their stimulation was antagonized by the ganglionic blocker, in combination with atropine. hexamethonium, These observations are consistent with previous studies on the sympathetic innervation of the gastrointestinal tract (15,16) that have shown that stimulation of the sympathetic nerves, with few exceptions, excites sphincteric and inhibits nonsphincteric structures. They are also supported by previous histologic studies showing that noradrenergic termi-
Figure
k 1.9
0.70
[ 28.2 t 6.8
n = Number of experiments.
the LES response
P”
53.0 + 13.0"
15
Bilateral adrenalectomy
Central end -___---_-
end
8. Tracing
illustrates the LES response to electrical stimulation (10 Hz, 10 V) of the peripheral end of the left gastric artery.
Black dot indicates the timing tion of the stimulus.
and dura-
the significance
C n
20-
Pb I
0.50
0.90
of the differences
in
nals are found in the circular muscle of the sphincter and that noradrenergic neurons are present outside the gastrointestinal tract (13). Stimulation of the splanchnic nerves contracts the LES due to direct action of norepinephrine released from postganglionic axon terminals on the circular smooth muscle (17). The action of the endogenously released norepinephrine during stimulation of the splanchnic nerves was in agreement with that of exogenous norepinephrine. In the cat, exogenous norepinephrine caused LES contraction by acting directly on the circular muscle because TTX does not prevent this action (12). The LES response during excitation of this reflex arc is not mediated by stimulation of the adrenal glands. This possibility was entertained because glucagon caused LES contraction by releasing epinephrine or norepinephrine from the adrenal glands (12). Bilateral adrenalectomy blocked the effect of glucagon. In spite of the anatomic proximity of the sympathetic branches arising from the celiac ganglia to the adrenal glands, bilateral adrenalectomy did not impair the LES response to sympathetic stimulation. Basal LES tone is not dependent on the integrity of this reflex arc. Basal LES pressures remain un-
w 60-
II v) o!!i
---
40 Hz
60 SECONDS
NEURAL CONTROL OF THE CAT LES
April 1982
Table
6.
Effect of Section of Extrinsic Lower Esophageal Sphincter
‘K--v-l0
Cervical vagus nerves Thoracic vagus nerves Spinal cord Splanchnic nerves Periarterial sympathetics of the LGA
40
20
FREQUENCY
Figure 9. Frequency-response studies of electrical stimulation (5-40 Hz) of the peripheral end of the left gastric artery. Values are mean ? SE.
changed after interruption of this excitatory reflex at the cervical vagus nerves or at the spinal cord. These observations are supported by the finding that hexamethonium and atropine, which effectively eliminate the contribution of preganglionic fibers of this reflex, do not affect basal LES pressures. It is still possible, however, that the postganglionic adrenergic neurons of the sympathetic pathway may contribute to the genesis of LES tone. Complete LES sympathectomy was not achieved by section of the left splanchnic nerves or of the periarterial sympathetics of the LGA. The finding that phentolamine, an cY-adrenergic antagonist, caused a moderate but consistent reduction of basal LES pressures supports this assumption. It was further confirmed by the studies with TTX. These phentolamine or TTX effects are not related to a concomitant fall in arterial blood pressure. A similar blood pressure fall due to arterial bleeding produced only a transient reduction
Nerves on Basal Pressures
Control lmmHrrl
n 9 3 6 7 7
30.8 36.4 34.0 48.3 52.4
687
Nerve section
-+ 4.8’ % 3.8 C 6.4 + 3.9 ‘- 4.7
37.2 37.9 30.2 45.6 51.3
t c t 2 ?
Pb
4.2 0.20 4.7 0.50 5.1 0.60 2.4 0.40 4.7 0.80
n = Number of experiments. ’ Values are mean ? SE of absolute LES pressures. b Paired Student’s t-test comparing pressures before and 30 min after nerve section.
in LES pressures (18). Alternative explanations, however, are possible. As in the opossum, the LES circular muscle may be primarily responsible for its tone, but it is regulated by a balance of excitatory and inhibitory neural influences. It is conceivable that phentolamine antagonizes the excitatory nerves enhancing the influence of TTX resistent inhibitory nerves (19). These studies do not support the existence of a postganglionic cholinergic neuron within this reflex arc as effective doses of atropine alone did not
Table
7.
Effect of Pharmacologic Antagonists on Basal Lower Esophageal Sphincter Pressures
Control
n
Basal pressures (nunHal
% Change
)
6
( “,;:r 1 ::;)
-28.3”
)
7
( ::::
1
9
(;;:;:I;)
1
9
( :;I’, : :I:
)
-12.2
]
8
[ :I:;;:;;
)
--25.8
- -- Pb 0.001
Tetrodotoxin (12 pg/kg)
Table
-_..
5.
_.
Effect of Pharmacologic Antagonists on the Lower Esophageal Sphincter Response to Electrical Stimulation of Periarteriaf Sympathetics of Left Gastric Artery --LGA stimulation n
__. _.-_.._IControl
_-_- __.~__..
AP (mmHg) - ._-_-
Atropine (30 wg/kg) Control
6
6
Hexamethonium and atropine
I ““‘”
I
0.001
6.6
TTX (12 pg/kg) Control
0.001 7
( 1°.Y
)
--8.3
f5.5
1.7 )
I
n = Number of experiments. a Mean 2 SE of LES pressure changes above basal pressures. b Paired Student’s t-test was applied to the LES response to LGA stimulation (10 Hz, 10 V) before and 30 min after the administration of each pharmacologic antagonist.
0.10
0.50
Hexamethonium
Control
Control
: ‘:I;
Control
Hexamethonium and atropine
Phentolamine (1 mg/kgl
Control
0.40
0.001
Phentolamine (1 mglkg) Control
0.30
Propranolol (1 mg/kgl
n = Number of experiments. a Numbers are mean 2 SE of absolute LES pressures. b Paired Student’s t-test comparing pressures before and after the administration of pharmacologic antagonists. c Values represent the percent of pressure change induced by drugs; control pressures are counted as 100%.
668
GASTROENTEROLOGY Vol.
BEHAR ET AL.
antagonize the LES contraction in response to vagal or sympathetic stimulation even though they effectively blocked the maximal dose of exogenous bethanechol. Only after the addition of the ganglionic blocker, hexamethonium, did the importance of the cholinergic receptors become apparent. These receptors appeared to be present at the celiac ganglion cells, as the stimulation of the postganglionic periarterial fibers of the LGA was not affected by these antagonists. These studies do not support the hypothesis that a postganglionic cholinergic neuron participates in the genesis of basal LES tone. There is, however, indirect evidence of the existence of such a neuron in the cat because the LES contraction induced by 5-hydroxytryptamine is partially blocked by atropine (Behar J: unpublished observation). Other in vitro (20) and in vivo (21,X?) studies on the cat LES have suggested the presence of postganglionic cholinergic neurons contributing to the genesis of sphincter tone. Different methodology and experimental design may explain these discrepancies. The anchored manometric probe used in this study allowed us to obtain continuous and accurate LES measurements throughout the experiment. We have observed transient reductions in LES pressures 5 or 10 min after the administration of atropine; LES pressures, however, returned to baseline levels while there still was an effective blockade of a maximal dose of bethanechol. Lower esophageal sphincter relaxation was never observed during stimulation of this reflex arc even after blocking excitatory receptors. Sphincter inhibition during stimulation of the central end of the splanchnic nerves has been recently shown in the cat (22). This study differed from ours in at least two significant aspects: [a) LES pressures measured were only lo-15 mmHg, whereas in our laboratory we found pressures of 44 ? 12 mmHg (mean ? SE in 412 animals). Other studies in the same animal species have shown results similar to ours (23). This pressure difference cannot be easily explained because it cannot be accounted for by either flow rate or by diameter of the catheter. (b) The manometric catheter was anchored at the LES by placing a small arterial clip, which in our preliminary experiments was not sufficient to anchor the pressure sensor firmly within the LES and prevent movements of the catheter. In conclusion, the present studies show the existence of an excitatory reflex arc with an afferent vagal and an efferent sympathetic pathway to the cat LES. It consists of preganglionic cholinergic fibers and postganglionic adrenergic neurons that stimulate a-receptors. Although its physiologic role remains unclear, it is possible that the postganglionic adrenergic neurons may contribute to the genesis of
basal LES tone or to its regulation tonic neural inhibitory influences.
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