Sympathetic Control of Lower Esophageal
Sphincter Function in the Cat: ACTION OF
DIRECT CERVICAL AND SPLANCHNIC NERVE
STIMULATION
Jacques Fournet, … , William J. Snape Jr., Sidney Cohen
J Clin Invest. 1979;63(4):562-570. https://doi.org/10.1172/JCI109337.
The purpose of this study was to determine the effect of direct stimulation of the sympathetic nerves on the lower esophageal sphincter (LES) in the anesthetized cat. Neither unilateral nor bilateral cervical sympathectomy, or splanchnicectomy significantly modified basal LES pressure in animals with intact vagi, or animals having undergone bilateral cervical
vagotomy. Electrical stimulation of the cut, peripheral, cervical sympathetic trunk increased mean arterial blood pressure, but had no effect on LES pressure or LES relaxation as
induced by vagal stimulation. Stimulation of the central end of the cervical sympathetic trunk had no effect on LES pressure. Stimulation of the central end of the cut splanchnic nerve produced a decrease in LES pressure with a maximal response of 69.1±16.0%
(mean±SEM). This inhibitory response was not modified by either propranolol or bilateral cervical vagotomy. Stimulation of the peripheral end of the cut, greater splanchnic nerve gave an increase in LES pressure with a maximal response of 38.2±7.19 mm Hg.
Guanethidine, in the presence or absence of the adrenal glands, significantly augmented this excitatory response. This response was also slightly increased by phentolamine alone at 10 V, 1 Hz, but was not altered by propranolol. The excitatory response was completely antagonized by atropine or by trimethaphan camsylate. Stimulation of the peripheral end of the splanchnic nerve inhibited LES relaxation as induced by […]
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Sympathetic Control of
Lower
Esophageal Sphincter
Function
in
the Cat
ACTION OF DIRECT CERVICAL AND SPLANCHNIC
NERVE STIMULATION
JACQUES FOURNET, WILLIAM J. SNAPE, JR., and SIDNEY COHEN, Gastrointestinal Section of the Department ofMedicine, University of Pennsylvaniaatthe Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104
A B S T R A C T The purpose ofthis study was to de-termine the effect of direct stimulation of the
sympa-theticnerves onthe lower esophageal sphincter(LES)
intheanesthetized cat.Neither unilateralnorbilateral
cervical sympathectomy, or splanchnicectomy
signif-icantly modified basal LES pressure in animals with
intact vagi, oranimals having undergone bilateral
cer-vical vagotomy. Electrical stimulation of the cut,
pe-ripheral, cervical sympathetic trunk increased mean
arterial bloodpressure,but hadnoeffecton LES
pres-sure or LESrelaxationasinduced
by vagal
stimulation.Stimulation of the central end of the cervical
sympa-thetic trunk hadnoeffecton LESpressure.Stimulation
of the central end of thecutsplanchnicnerveproduced
adecreaseinLESpressure withamaximalresponseof
69.1 ±16.0%
(mean±
SEM). Thisinhibitory
response was notmodifiedby eitherpropranololorbilateralcer-vical vagotomy. Stimulation ofthe peripheral end of
the cut, greater
splanchnic
nerve gavean increase in LES pressure with a maximal response of38.2 + 7.19mm Hg. Guanethidine, in the presence or absence of the adrenal
glands,
significantly augmented
thisexcita-tory response.Thisresponsewasalso
slightly
increased by phentolaminealoneat 10V, 1Hz, butwas notalteredby propranolol.The excitatory responsewascompletely
antagonized byatropineorbytrimethaphan camsylate.
Stimulation of the peripheral end of the splanchnic
nerve inhibited LES relaxation as induced by vagal
stimulation. The results of this study suggest that: (a)
the LES in the cat is notaffected by either central or
Dr. Foumetis supported by funds from the University of
Grenoble, Grenoble, France.
Received forpublication24July1978andinrevisedform
8December1978.
562
peripheral stimulation of the cervical sympathetic trunk; (b) the central portion of the splanchnic nerve carries an afferent inhibitory response to the LES through yet unknown pathways; (c) the peripheral splanchnic nerve carries an atropine-sensitive excita-toryresponse to the LES; and (d) the splanchnic nerves
may modulate LES relaxation as induced by vagal
stimulation.
INTRODUCTION
In recent studies, the role of the vagal nerves in the control of loweresophageal sphincter(LES)' function
has been emphasized. The vagi serve a major role in
the initiation of LES relaxation during swallowing (1, 2). Excitatory cholinergic pathways in the vagi have
beendifficulttodemonstrate in the opossum, but may
be present in the cat (3, 4). Our understanding of the influence of vagalfunctionon the LES had developedl
through studies designed to inhibit or stimulate the
vagi directly (1, 2). These studies allowed the
identi-fication of neural pathways ofthe nonadrenergic
in-hibitory or purinergic type that were not previously
recognized during earlier studies, in which only agonists or antagonists were parenterally administered (5). At the present time, our understanding of the sympathetic control of LES function is based upon studies in which alpha or beta adrenergic agonists or antagonists were given (6, 7). Recent studies using accurate measuresof LES function have not been
per-formed with direct stimulation of the sympathetic
in-nervation to the LES (8, 9).
'Abbreviationusedin this paper: LES, loweresophageal sphincter.
The purpose of this study is threefold: first, to de-termine the effect of direct stimulation or section of
the cervical sympathetic orgreatersplanchnic nerves;
second, to identifv the nieural transmitter involved in
mediating these changes in LES pressure; and third,
to
study
the interaction between the sympathetic and vagal pathways involved in the regulation of LES function.NIETHODS
Studies wereperformedlin 34adultcats of eithersex, weigh-ing between 2.0anid4.5kg.Eachanimalwas fasted for 12-18 h before the manometric studyand was initially anesthetized with sodium pentobarbital (50 mg/kg, i.p.) or ketamine (10
mg/kg). Intravenous anesthesia was given as needed into a
catheterin the femoral vein (pentobarbital, 10 mg; or keta-mine, 2.0 mg/kg). Afterinductionof the anesthesia, a tracheot-omywasperformed for assistedventilation with a respirator. A cannula inserted into the femoral artery was filled with heparin and connected to an external transducer (Statham P231A,StathamInstruments, Inc.,Oxnard, Calif.)for constant blood pressure monitoring. The rectal temperature of the animalwasmaintainedat aconstantlevelwith aheat lamp.
The neck was explored through a midline incision. The
right andleft vago-sympathetictrunks wereidentifiedwithin
the carotid sheath between the internal jugular vein and
the carotid artery. The sympathetic and vagal trunks were than isolated during microscopic examination and secured with two loops ofumbilicaltape. Thesympathetictrunk was characterized as being smaller than the cervical vagus. The sympathetictrunk hadprominent gangliaand rami
coimmlluIni-cantesgoing to thespinalcord. Nerve identity was confirmed
at postmortem examiniation by determining continuity with
thethroacic sympathetictrunk.
Theabdomen wasopenedthroughamidlineinlcisioIn.The right or left greater splanchnic nerve was iclentified and isolatedas ittransversedthediaphragm before itsentryinto
the celiac ganglion. The trunk was then secured with two
loops of umbilical tape. Nerve identity was againconfirmed at postmortem examination by identifying its proximal
con-nection to the thoracic svmpathetic trunk and its distal
connection tothe celiacganglion.
Esophageal manometric studies were performed using a tubeassemblyconsistingofthreepolyvinylcatheters (1.4 mm inside diameter) vith side orifices ofsimilarsize spaced at 5-cmintervals. Each catheter wascontinuously infused with distilled water at arate of1.2 ml/min. This rate of infusion gave a>200 mm Hg/s riseinpressure upon occlusion of the recordingorifice. Theintraluminalpressures weretransmitted to external transducers with outputs graphed on aBeckmnani
rectilinear ink writingrecorder(Beckman Instruments, Inc.,
Fullertoni,Calif.).
Ineachanimal,therecordingcatheter waspassed through the mouth into the stomach. The catheter was withdrawn while recording pressure at 0.5-cm intervals for a 1.0-min period. Upon completion ofthe pull through, the recording tubewasanchored with the middle orifice intheLES atthe point ofmaximum recorded pressure. After thepullthrough, the tube was secured at the jaw with tape, and within the abdomen with small arterial clips fixed at the distal esoph-agus and mostproximalstomach. This arrangementprevented axial movement of the LES in relation to the tube orifice. This was verified using a separate tube with axial openings at 1.0-cm intervals. The LES high pressure remained at the
desired catheter opening. Also, manual orad movement of
the esophagus did not displace the LES from the recording orifice.
LES pressure wasrecorded as millimetersofmercury,with gastric pressure used as a zero reference; the values were obtained as the midrespiratory value. The results were ex-pressed as a percentage of change inthe sphincter pressure for the sphincterrelaxation, and as absolute values for the excitatory motor responses. The percentage by which LES pressure decreased duringswallowing was calculated as
rest-ing LES pressure minus the nadir of LES pressure during
swallowing, divided by resting LES pressure times 100. The
stomach was intermittently aspirated to prevent gastric
distension.
After recording the basal LES pressure, the right or left cervical sympathetic trunk was sectioned at a site 1.0 cm proxi-mal to the superior cervicalganglion. LES pressure was con-tinuously recorded during all procedures. After 30 min, the other cervical sympathetictrunkwassectioned. A similar pro-cedure was usedforthesplanchnicnerve studies. The greater splanchnic nerves were sectioned at a site 1.0 cm proximal totheceliac ganglion.
Electrical stimulation ofthecervicalsympathetic trunk, the cervicalvagi, or the greatersplanchnicnerves, wasperformed
with a platinum wire bipolar electrode (0.04 mm diameter)
which was mounted in polyvinyl tubing (11.0 mm length,
5.0 mm diameter) and was sealed with bonded adhesive.
The distance between stimulating points was 5.0 mm. The distal or proximal ends ofthe nerves were placed into the trough-shaped electrode, covered with cotton, and saturated with mineral oil. Care was taken to prevent currentleakage
by elevating the nerve from adjacent tissues with cotton
pledgets. An electrical stimulus was delivered with a Grass stimulator(Model 88, with stimulus isolation unit SIU 5, Grass Instrument Co., Quincy, Mass.). Square wave pulsesof1.0-ms
stimulus duration, were delivered at a frequency range of
0.50-50 Hz and a voltage range of 5 or 10 V. The length
of the stimulus train was 5 s. The trains were separated by intervals of at least 5 min. A special procedure was carried outfor the simultaneous stimulation of the cervical vagaltrunk and thesympatheticnerves.The lengthofthe stimulustrain was 15 s, either for the cervicalsympathetic trunk or the greater splanchnicnerves; simultaneous vagal stimulation was per-formed 10 s after the onset of sympathetic stimulation. Re-sponses tovagal orsplanchnicnerve stimulation began with the onsetofnerve stimulationand persisted for the duration of stimulation,returning to basal values upon the termination ofthe stimulus.
Drugs were administered through an intravenous catheter
into thefemoral vein. The agents were given as single intra-venous boluses over 60 s or by continuous infusion. Normal salinewasusedtoflush the catheterafter each drug
adminis-tration. The LES response to splanchnic nerve stimulation
was measured in the same animals before and after administra-tionof the different antagonists. A period of 60minfor stabili-zation tobase-line levelswasobserved after the administration of each pharmacological agent. 1.5 mg/kg phentolamine
(Regitine, CIBA-Geigy Corporation, Summit, N. J.) was
chosen as the dose thatantagonized themaximal LES response to phenylephrine at 24.0
jig/kg.
1.0 mg/kg propranolol (In-deral, Ayerst Laboratories, New York) was given as the dose thatantagonized the maximal LES response to 40 ,ug/kg iso-proterenol. 30.0 ,ug/kg atropinesulfate (The Vitarine Co., Inc., New York) was selected as the dosage that antagonizedcom-pletely the maximal LES response to 5.0 mg bethanechol.
Additional studies with 10.0 and 100.0 ,Ag/kgatropine were performed. Trimethaphan camsylate (Arfonad, Roche Labora-tories, Div.Hoffmann-LaRocheInc., Nutley, N. J.), 4.0mg/min, wasgiven at a dosage that inhibited the maximal blood
sureresponse duringsplanchnic nerve stimulation or the LES response to nicotine sulfate (50.0 ,ug/kg). 5.0-10.0 mg/kg guanethidine (Ismelinf, CIBA-Geigy Corporation) was given at dosages previously reported to be effective in lowering blood pressure and blocking adrenergic neural function. Guanethidine effect was evaluated at 10-60 min after its adminstration. Alone, guanethidinegave significant reduction in blood pressure in all animals.
To eliminate the effectofpreganglionic splanchnic nerve stimulation upon the adrenal glands,agroupof animals were also studied after bilateral adrenalectomy.
Statistical analysis was performed with the paired t test forobservations made in a minimum ofseven animals. All records were read in a blinded fashion without knowledge of the nervestimulationorofthedrug being employed.
RESULTS
BasalLES pressure in response tocervicalor greater
splanchnic nerve section. Cervical sympathetic sec-tion (unilateral or bilateral) at a site proximal to the
cervical ganglion failedto significantly alter LES pres-sure. Greater splanchnic nerve section (unilateral or
bilateral) at a siteproximal to the celiac ganglion also did not significantly change the basal LES pressure.
The control LES pressure was 15.66± 1.41 mm Hg (mean±SEM) as compared with 16.02±1.28 mm Hg
after bilateral cervical sympathectomy (P>0.05) and 18.00±2.10 mm Hg after bilateral splanchnic nerve section (P >0.05). LES pressures were similar with
either pentobarbital orketamine.
Cervicalvagotomy(unilateralorbilateral) also failed
to alterthe basalLEStone. The LES pressure incontrol
animals was 15.54+2.06 mm Hg as compared with
15.37±2.15 mm Hg after unilateral cervical vagotomy (P >0.05), and 15.64±2.31 mm Hg after bilateral cer-vical vagotomy (P±0.05). The basal LES pressure was
also obtained inanimals undergoing bilateral cervical
vagotomy with either unilateral or bilateral cervical
sympathetic or greater splanchnic nerve sectioning.
Vagotomy plus sympathectomy also did not lead to
significant alterations in basal LES tone (P >0.05).
Effect of
peripheral
cervicalsympathetic
nervestim-ulationon LESpressure,vagal-inducedLESrelaxation
and blood pressure. Fig. 1 shows the percent
de-creaseinLESpressure(above)and thechangeinmean
blood pressureinmm Hg(below) fora fullfrequency
rangeat 5 V(left) and10 V(right).Cervical sympathetic
nerve stimulation did not alter LES pressure at either
5 or 10V over the entire frequency range. Cervical
sympatheticnerve stimulation did giveasmallincrease
inmean arterial blood pressure. Cervicalvagal
stimu-lationgaveadecreaseinLESpressure andmeanblood pressure. Simultaneous stimulation ofthecervicalvagus and cervical sympathetic trunks did not alter either the percentage decrease inLES pressureorthe mean
decrease in arterial blood pressure at either voltage overthe full frequency range.
Effect of stimulation of the central portion ofthe
100
80
(I)
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n)
cx
0
50
20
0
0w +20
4Al
(L +10
E - 10
E -20
*-3
0
o, I.
- I (>
0-?,1
....4... ...//... -4
*v
--o V's
4. s
,
1 0 30' 50 1510 20 30 50Frequency Of Stimulation (Hz) (5 Volts) (10Volts)
FIGURE 1 Effect ofelectrical stimulationof thedistalend of the sectioned cervical sympathetic trunks on LES pressure (above), and on mean blood pressure (below). Sympathetic stimulation(s) producednoeffect on sphincter pressureover
the entire frequency range at 5 and 10 V; it induced a
slightincreaseinmeanbloodpressure. Peripheral vagal stim-ulation (V) induceda frequency-related sphincter relaxation and adecrease inblood pressure. Simultaneous stimulation ofthe sympathetic nerve (V + S) did not modify sphincter relaxationand blood pressure responses asinducedbyvagal stimulation.
cervicalsympatheticorsplanchnicnerves on LES
pres-sure; action ofantagonist agents. Fig. 2 shows the
effect of electrical stimulation ofthe central end of
the cut cervical
sympathttic
or greater splanchnicnerves. Data isexpressedas apercent decrease in LES
6L
a-(n w -J
c
0)
0
°OOr
80
60
40
20
*CONTROL (0-4*)
BILATERAL CERVICALVAGOTOMY(0...0 SPLANCHNIC
d;r r 7
- PROPRANOLOL(Img/kg) (--*4
Y 0 -0CERVICALSYMPATHETIC(0-O)
1 5 10 20 30 50
Frequency Of Stimulation (H!)
FIGURE 2 Effect ofelectrical stimulationofthecentralend of
the sectioned cervical sympathetic or splanchnic nerve on
LESpressure.Electrical stimulation at 10 V wasappliedover an entire frequency range. Stimulation of the central end of the cut cervicalsympatheticnerve gave no change insphincter pressure. Stimulation of thecentralend of the cutsplanchnic nerve produced a decrease in sphincter pressure. Bilateral cervicalvagotomyorpropranololdid notmodifythisresponse.
pressure at 10Voverthefullfrequencyrange. Central
cervical sympathetic nerve stimulation did not alter
LES pressure at any point. Stimulation of the central portion of thegreater splanchnicnervegaveadecrease in LES pressure; maximum inhibition of 69.1±16.0% was noted at a frequency of 10Hz.
The inhibition inLESpressure noted during central stimulation ofthe cutsplanchnicnerve was next stud-ied during bilateral cervical vagotomy, or after the administration of propranolol (1.0 mg/kg, i.v.). This dose ofpropranolol was selected as the lowest dose whichproducedcompleteantagonism of themaximum inhibitory effect of isoproterenol (4.0
/Lg/kg,
i.v.) onLES pressure. Change inLES pressure wassimilarat
all frequencies (P> 0.05) after either vagotomy or
propranolol.
Inadditional studies, not shown here, it was
demon-stratedthatbilateralcervicalsympathectomyorcutting
of the contralateral splanchnicnervealso failedtoalter
the response to central splanchnic nerve stimulation.
Effect of stimulation of the
peripheral
portion ofthesplanchnic nerveonLES pressure. Fig.3Ashows
the effect of electrical stimulation of the peripheral
end of the greater splanchnic nerve at 5, 7, and 10V
over a full frequency range. Peripheral splanchnic
nerve stimulation gave an increase in LES pressure.
These changes in LES pressure werevoltage and
fre-quencydependent. The LESpressure changesat5V
were not statistically significant. The LES pressure
changesat 7 Vweresignificantatfrequencies of10and 20 Hz. At10V, theLESpressurechangeswere
signif-icant at 5, 10, 20, and 30 Hz. These changes in LES pressure wereobservedwithbothintact and cutvagal
trunks.
Fig. 3Bshows the time course of the LES response
to right splanchnic nerve stimulation of 15- or 5-s
duration. LES pressure rises rapidly but falls slowly
aftertermination of the stimulus.
Effect of ganglionic blockade upon the excitatory
effect of
peripheral
splanchnic nerve stimulation onLES pressure. Fig. 4 shows the effect ofganglionic
blockade with trimethaphan camsylate (4.0 mg/min,
i.v.) upon LES pressure and mean arterial blood
pres-sure during stiniulation of the peripheral end ofthe
greatersplanchnic nerve. Trimethaphan camsylate was selected at adosage thatwouldantagonize theincrease inblood pressure during splanchnic nerve stimulation.
Trimethaphan camsylate completely antagonized the
increase in LES pressure over the entire frequency range of direct peripheral splanchnic nerve stimulation.
Effectofguanethidineand bilateral adrenalectomy
upon the excitatory effect of peripheral splanchnic
nerve stimulation onLESpressure and blood pressure. Fig. 5shows the effect of stimulation of the peripheral end of thesplanchnicnerve inthe presence of guaneth-idine (5.0 or 10.0 mg/kg, i.v.) alone, and guanethidine
I
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0)
-j
tn
a)
CL
Ji
40F
A
A410v
.---
,,-
,7V
.. 11-1-,,---.
5
V
30
-20F
1o0
0 5 10
20
30
50
Frequency
Of
Stimulation
(Hz)
B
50- i30s--- 50 30
401-
40-E 20-J E2o
-E E
10 10
0 10V. 0Hi 0 IOV.10Hz
FIGURE3 (A) Effect ofelectrical stimulation of the periph-eraleyidof the cut greatersplanchnicinerveat5, 7,aind10Vover a frequency range of 1-50 Hz. Electrical stimulation pro-ducedanincrease in LESpressure. Thechangein LES pres-sure was significantat 7 and 10V(indicatedby an asterisk).
(B) Time course of LES response to right splanchnic nerve
stimulation at 10 V, 10 Hz fora stimulus train at 15 s (left)
and 5 s (right). Pressure rose rapidly, but fell slowly upon termination of the stimulus.
plus bilateral adrenalectomy. Alone, guanethidine
significantly augmentedthe excitatoryeffect of
stimu-lation ofthe peripheral end of the splanchnic nerve
at frequencies of 5, 10, 20, 30, and 50 Hz on LES
pressure. Guanethidine alone also gave a slight aug-mentationofthe increase in mean arterial blood pres-sure.These changesinblood pressure were not signif-icant. Because guanethidine does not alter adrenal medullary activity during splanchnic nerve stimula-tion, animals were further studied after bilateral adre-nalectomy. After bilateral adrenalectomy, guanethidine continued to augment the LES pressure response to
peripheral splanchnicnervestimulation. The increase
in LES pressure was slightly higher than seen with guanethidine alone, but this difference was not signif-icant. Bilateral adrenalectomy in combination with
Sympathetic Controlof Lower Esophageal Sphincter Function
OL
E 40
E~~
30-CONTROL (@_)
TRIMETHAPHAN CAMSYLATE (4mg/min) (O--O)
L20
-w
I-J 0
~40[
8E
Ec~20S-a O-- 0_ 0_ -0-4 4O--V/--O
X 0 5 10 20 30 50
Frequency Of Stimulation
(Hz)
FIGURE4 Effect oftrimethaphan camsylateonthefrequency
response curve ofperipheral splanchnic nerve stimulation
upon LES pressure(above), andmeanbloodpressure(below),
at 10 V.Trimethaphan camsylate completely suppressed the
excitatory effecton both the sphincteric and bloodpressure responses.
-80 E
60-
40-cn
20
-l
0
E
40°
I0' 20
*01
&10
E * GUANETHIDINE +BILATERAL ADRENALECTOMY (00)
P ti GUANETHIDINE (5 nd 0mOg/kg) ( 0)
P CONTROL (0-10
*P' 0.05
.. I GUANETHIDINE- - (5and10mg/kg)
J~A---i-- -4 CONTROL(O_)
GUANETHIDINE (5,ondOmg/kg)+
- qF--- - BILATERALADNIENALECTOMY (0--O) 5 10 20 30 50
Frequency Of Stimulation (Hz)
FIGURE5 Effect of guanethidineonthefrequencyresponse
curve of peripheral splanchnic nervestimulation upon LES pressure(above), andmean bloodpressure (below),at 10 V.
Guanethidine (5 and 10mg/kg) increased theexcitatoryeffect ofsplanchnicnerve stimulation onLES andbloodpressure.
Guanethidine (5 and 10 mg/kg) plus bilateral adrenalectomy abolishedthechangeinbloodpressure,but continuedto
aug-ment the LES pressure response to peripheral splanchnic
nervestimulation.
guanethidine completely abolished the changes in mean blood pressure during peripheral splanchnic nerve stimulation. Adrenalectomy alone reduced the
bloodpressurebutnotthe LES response to splanchnic
stimulation.
These observations suggested that the inhibition of
catecholamine release byguanethidine augmentedthe
increase in LES pressure during peripheral splanchnic nerve stimulation. These changes in LES pressure
could be dissociated fromthealterationsinblood
pres-sure noted duringnerve stimulation.
Effect of alpha adrenergic antagonism with
phentol-amine upontheexcitatoryeffect ofperipheral
splanch-nic nervestimulationon LESpressure and meanblood pressure. Fig. 6showsthe effect of phentolamine (1.5
mg/kg, i.v.) upon the excitatory effect of peripheral
splanchnic nerve stimulation on LES pressure and
mean blood pressure. Phentolamine was selected at
the lowest dosage that would completely antagonize
the maximal excitatory action of phenylephrine (24.0
p,g/kg, i.v.) on the LES. Phentolamine augmented the
effect on the LES response to peripheral splanchnic
nerve stimulation at 10 V, 1 Hz. There was no effect
at other stimulus parameters. Phentolamine did
com-pletelyantagonizethe increase in mean blood pressure
noted during splanchnic nerve stimulation.
Thus, alpha adrenergic antagonism with
phentol-amine, at adosage sufficient to block the maximal ex-citatory action ofphenylephrine on the LES and the
PHENTOLAMINE (1.5Img/kg)
CONTROL
CONTROL
l----_- _-s..--- -4----4 PHENTOLAMINE (1.5mg/kg)
0 0.5 1 3 5 10 20 50
Frequency Of Stimulation (Hz)
FIGURE 6 Effect of phentolamineonthe frequencyresponse
curve of peripheral splanchnic nerve stimulationupon LES pressure(above),andmeanbloodpressure (below),at 10 V.
Phentolamine increased theLESpressureresponseto periph-eralsplanchnicnervestimulationat 1 Hz,but hadno
signif-icant effect at the other frequencies. Phentolamine did
completelyantagonize the increase in meanblood pressure
notedduring splanchnicnervestimulation.
f
X0
E
408oE
mc,,
effect of splanchnic nerve stimulation on blood pres-sure, failed to alter the excitatory action of nerve
stimulation on LES pressure.
Effect of combined alpha and beta adrenergic
an-tagonism upon the excitatory effect of peripheral
splanchnicnerve stimulation on LESpressure. Fig.7
shows the effect of propranolol (1.0 mg/kg, i.v.) alone,
andpropranolol plus phentolamineupon theexcitatory
effect of peripheral splanchnic nerve stimulation.
Neitherpropranolol alone,norpropranolol plus
phentol-amine significantly altered the LES response to
splanchnic nerve stimulation. Blood pressure
re-sponses wereinhibitedduring propranolol and
phentol-amine administration, butnot with propranolol alone.
Effect of atropine sulfate upon the excitatory effect
of peripheral splanchnic nerve stimulation on LES
pressure and mean blood pressure. Fig. 8 shows the
effect of intravenous atropine sulfate at doses of 10,
30, and 100 ,ug/kg upon the LES pressure and blood
pressure responses to peripheral splanchnic nerve
stimulation. Each dose ofatropine reduced the LES
pressure response tosplanchnicnervestimulation. The
changes in response to atropine at 10
,ug/kg
were notsignificant. Both higher doses ofatropine, 30 and 100
I
E
E
1-0~
CL (n
w IJ
O.-40r
m E 40
E
X0
020
C
20
a-
O. .o... 0..
o
I.. I , A--.
0 5
10
20
30
50
Frequency Of Stimulation
(Hi)
FIGURE7 Effect ofpropranolol onthe frequency response curveofperipheral splanchnicnerve stimulationonLES pres-sure (above),and meanblood pressure (below),at.10V. Pro-pranololdidnotchange the sphincterpressureorblood pres-sureresponsestosplanchnicnervestimulation. The
comhibina-tion of the adrenergic antagonists, phentolamine and
propranolol,wasalsoineffective.
I
E E 40
1-a)
U) 30
10
20
o E
a.
20
0-..
20
m0
0 a)
-J > Control
Atropine (10 9Lg/kg)
f--- "-1Atropine (30 ,g/ kg)
.o..-...-.o.--.oAtropine (100 4g/kg)
> Control
-t--"-
~i
Atropine (30,4g/kg)5 10 20 30 t0
Frequency Of Stimulation (Hz)
FIGURE 8 Effect of atropine sulfate on the frequency
re-sponse curve ofperipheral splanchnic nerve stimnulation on LES pressure (above), and mean blood pressure (below), at 10 V. At a dose of 10.0 ,ug/kg, atropine did not signif-icantly decreasethesphincterpressure response tosplanchnic nerve stimulation. At a dose of 30.0
,gWkg,
atropinesignif-icantly decreased this response. A dose of 100.0 ,Lg/kg atropinecompletely! suppressedthe sphincteric responsefor several hours. No significant change in hloocl pressure re-sponses was noted at each dose ofatropine.
,ug/kg,
significantly
reclticed or abolishedtheinereatse
in LES pressure dIurinig splaciehiuic nerve
stimltulationi.
The complete antagonismii ofthe LES response during
atropine at 100 Ag/kg lasted for several hours. At all
doses atropine failedto
significantly
alterthe inereasein meean bloodpressure during
splanchnici
nervestimll-ulation. Identical effects ofatropine on the LES re-sponse to
splanchiuic stimulationv
were observed with intact or cut vagi.Effect of peripheral
splanchn
ic nerve stimulation
upon
LESrelaxation
asinducedby
vagalstitmiulationi.
Fig. 9 shows the effect of peripheral
splanehiic nerve
stimulation
upon LES relaxation as induced by vagalstimulation. Results are shown for vagal
stimiiulationi
at 10 Vovera full
frecquency
range.Splanchiuic
stiniu-lation was tested at 10V, 20 Hz (left)
aind
10 V, 10 Hz(right). Splanchnic
stimulation
at eitherfrecquency
significantly reducedsubimcaximiail and milaximiail
inhibi-tory responses to vagal
stimulaltioni.
Theseresuilts
shouldbecontrasted with thefindinigs
in Fig. 1, where cervicalsymipathetic stimulaltion
wasshowin
tohaven1o
effect on LES relaxation asiniduicedl
byvatgal
stimulation.
Effect of
atropitne oni
theactiotn
of peripheralsplanchnic stimulation
oni
LESrelaxation
asiniducedc
a .-- i Control ,. Control 80
-j
60-.e..i Splanchnic
40
o 20 (IOV, 20 Hi
Q .=P'~~~~~~~~~~005
__.___. _. _..
5 10 20 30 50 I510 20 3 '50
Frequency Of Stimulation (Hz)
FIGURE 9 Effect of peripheralsplanchnicnervestimulation duringLESrelaxation as induced by vagal stimulation. Elec-trical vagal stimulation at 10Vinduced LES pressure relaxa-tion. Simultaneous stimulation ofthe peripheral end of the splanchnic nerve at the parameters of stimulationofmaximal effect on the sphincter (10V, 10 Hz or 10 V, 20 Hz) signif-icantly decreased the submaximal and maximal inhibitory sphinctericresponses to vagal stimnulation.
by vagal stimulation. Fig. 10shows the effect of atro-pine sulfate on splanchnic stinmulation during LES
re-laxation as induced by vagal stimulation. Atropine sulfate was given atthe dosage (30
Ag/kg,
i.v.) showntoantagonize the excitatory effectofsplanchnic nerve
stimulation on LES pressure (Fig. 8). Atropine
com-pletely antagonized the inhibitory effect of splanchnic nerve stimulation uponLES relaxation. After atropine, LES relaxation was similar to control values at each frequency.
DISCUSSION
The results of these studies suggest several points. First, the cervical sympathetic nerves proximal to the
01) L.
U)
U)
lU)
L.
_o
o) w
a)
(I)
0)
a) 0N
100
80
60
40
20
Frequency
Of
Stimulation
(Hz)
FIGURE 10 Effectofatropine sulfateon splanchnic
stimula-tion during LES relaxationi as induced byvagal stimulation.
Atropine (30.0 jig/kg) abolished the antagonistic effect of splanichniicnervestimulation(10 V, 20 Hz)uponLESpresssure
relaxationl durinlg vagal stimulationi.
cervicalganglion have no effect on LES pressure when stimulated centrally or peripherally. Second, the cen-tral portion of the greater splanchnic nerve has an in-hibitoryeffecton LES pressurethatactsindependently
of the beta adrenergic receptor or central vagal path-ways. Third, the peripheral portion of the greater splanchnic nerve has an excitatory effect on LES pres-sure that is mediated through a ganglionic synapse (possibly celiac ganglion), and is sensitive to atropine but not to alpha or betaadrenergicantagonism.Fourth,
inhibition of catecholamine release by guanethidine
augments the atropine-sensitive, excitatory effect of
splanchnic nerve stimulation on LES pressure. Fifth,
the independent effects ofsympathetic nerve stimula-tion on LES pressure and blood pressure can be dis-sociated with specific antagonist agents. Sixth, bilateral cervical sympathetic or greater splanchnic nerve
sec-tioning has little effecton the basal LES pressure.
The role of the sympatheticneural innervation to the
LEShas been difficulttoascertain.Most previous
stud-iesusedspecific agonists or antagonists to ascertain the function of the alpha orbeta adrenergic receptors. In
the esophagus of the opossum, the cat, and man, the
alphaadrenergicreceptormediatesan excitatoryeffect,
whereas the betaadrenergic receptorproduces an
in-hibitory response (6, 7). Direct antagonism ofalpha
adrenergicactivity givesasmall decreaseinbasal LES
pressure.Betaadrenergicantagonists havenoapparent effect on basal sphincter tone (7). Destruction of the
adrenergic nerves with 6-hydroxydopamine leads to
hypersensitivity to exogenous adrenergic agents, but
givesonly a smalldecreaseinbasalLES pressure
with-outaffectingLESrelaxation inresponse to swallowing
(7). Based on studies of this type, suggestions have
been made concerning the role of the actual
sympa-thetic nerves in regulatingLES pressure and LES
re-laxation. This study indicates that observations based upon exogenous agonistadministration may bear little
relationship totheactual function of the nerves
them-selves, when stimulated or sectioned. A similar
phe-nomenon wasrecently described for thevagal
innerva-tion to the LES. It was suggested that the major vagal
neurotransmitterwas acetylcholineand that the vagus
carriedexcitatorycholinergic fibertotheLES(5).
How-ever, when the vagiwere cut intheneck,LESpressure was notreduced (10).Furthermore, directvagal
stimu-lation led to a nonadrenergic, noncholinergic (puring-ergic) relaxation in LES tone (3, 11). The absence of excitatory cholinergic innervation to the LES within the vagus has led to certain doubts that the LES
re-ceived any direct excitatorycholinergicinnervation(3). The reduction inLESpressure withatropineincertain animals and in man was unexplained(4, 12).
Thisstudysuggests that the greatersplanchnicnerve
carries an excitatory atropine-sensitive responsetothe
LES. This conclusion is based upon the observation
(L
thatatropine sulfate; but notphentolamine,
proprano-lol,or guanethidine; antagonizes the increase in LES
pressureduring the peripheralsplanchnic nerve
stimu-lation. It is uncertain whether the action ofatropine
is upon the muscarinic receptor in the muscle mem-brane, or whether atropine acts at the ganglion. It is
mostlikely thatthe action of atropine is at the muscle membrane.
Even atsmall doses, atropine suppressed the effect
ofsplanchnic nerve stimulation for several hours. At
those doses, atropine is a highly selective antagonist
ofmuscarinic agents on smooth muscle (13). Theaction of small doses of atropine makes the other possible
action ofthis drug lesstenable. Underappropriate
ex-perimental conditions and in high dose, atropine is
known tohaveanalpha sympatholytic effect(14).
Like-wise,atropine in high doses may antagonize the nico-tinicaction ofacetylcholine atthe autonomicganglion
(13). On the other hand, muscarinic antagonism at a
ganglionic synapsedoesnotusually abolisharesponse
as observedinthese studies. Itshould be
emphasized
thatganglionic blockade with trimethaphan camsylate
abolishedtheLES responseto splanchnic stimulation,
but this drug also antagonized the increase in blood
pressure as well. Atropine reduced the LES response
without altering blood pressure.
Cholinergic pathways in the sympathetic nerves
have been described in other studies with different
endorgan responses (15-20). However, thisisthe
ini-tial demonstration ofan excitatorycholinergicresponse
obtained during sympathetic nerve stimulation.
Usu-ally, mixed, cholinergic, and adrenergic responses are
described(18, 20).The neural pathway ofthis
choliner-gic response is not clear; the simplest explanations
would be the existence of cholinergic fibers within the
splanchnic nerve, or some connection with the
para-sympathetic nerves (15, 18). The excitatory action of
splanchnic nerve stimulation on the LESwas specific
for this nerve and was not observed during cervical
symnpathetic
nerve stimulation.Guanethidine in the presence or absence of the
ad-renal glands augmented the LES pressure response to
splanchnic nerve stimulation. This response was
inde-pendent of the drug's effect on blood pressure. The
action of guanethidine in
augmnenting
the excitatoryaction ofperipheral splanchnic nerve stimulation was
unexpected. Guanethidine is known to cause
adrener-gic neuroblockade, noradrenaline potentiation, and
sympathom
iimetic effects by release of noradrenaline (21, 22). Noradrenaline potentiation seems to be un-related to its action on adrenergic nerves (23). The increaseinthe sensitivity of tissues to circulatingcate-cholamineswassuggested
by
theincreased responseinblood pressure during splanchnic nerve stimulation. Thisresponse was suppressed after bilateral adrenalec-tomv. Guanethidine has no effect on the release of
catecholamines from the adrenal medulla (24).
Adre-nalectomy didnot modify the LES response to
guaneth-idine during splanchnic nerve stimulation, thus
sug-gesting that the effect on the LES was not a result of
potentiation of the action of released catecholamines. A possible sympathomimetic effect of guanethidine seems unlikely as no change in resting LES pressure wasnotedafterdrug injection. The action of
guanethi-dine does not seem to berelated to noradrenaline
po-tentiation or a sympathomimetic effect, as its effects
were notmodifiedby an alpha blocking agent,
phentol-amine. It ispossible thatguanethidine augmentedthe LESresponse tosplanchnic stimulation by the blockage ofa simultaneous inhibitory adrenergic response elic-itedduring splanchnic stimulation (25-27). However,
the demonstration that effective alpha, but not beta,
blockage only minimally augments the excitatory
re-sponse to splanchnicnervestimulation makesthis
pos-sible explanation less tenable. Therefore, the
mecha-nism by which
guanethidine
augmented the LESresponse to splanchnic nerve stimulation remains
undefined.
The splanchnic nerves, when stimulated at the
pe-ripheral end, also seemed capable of inhibiting LES
relaxation inresponse to directvagal stimulation. This
inhibitory responsewas not seenduring cervical
sym-pathetic nerve stimulation. The physiological role of
the splanchnic nerves in altering the LES responseto
vagal stimulationisunclear. Thisstudy doesnotdefine
the significance ofthis responseinhealthorindisease,
butsimply indicates thepresenceofaneural pathway
within the splanchnicnerves that can limit the ability
of the vagi to induce LES relaxation. This splanchnic
response was sensitive toatropine butnot totheother
neural antagonists. A similar reduction in LES
relaxa-tion as induced by vagal stimulation has been shown
duringintravenousdopamine infusion and during
sub-cutaneousaddition of bethanechol (28, 29). Thepresent
study suggests that the magnitude ofLES relaxation
may
be
reduced by splanchnic nerve stimulation aswell as bycertain drugs.
Stimulationof thecentral end of thesplanchnicnerve
gave a decrease in LES pressure. This response was
unchanged after bilateral cervical sympathetic or
bi-lateralvagal sectioning in the neck. Additionally, direct
stimulation ofthecervicalsympatheticnervealso failed toelicit this response. Thecentralsplanchnicresponse was notantagonizedby a seemingly effective doseofa beta adrenergic antagonist, propranolol. Also, section
of the contralateral splanchnic nerve failed to abolish this inhibitory response. These results would suggest that the inhibitory response was transmittedby a
non-adrenergic neurotransmitter thatis carried tothe LES
throughunknownpathways.It ispossiblethat the
cen-tral portion of the splanchnic nerve connects with
in-hibitory pathwaysinthe vagus nerveswithin thethorax.
This mlay occur without connection to thebrain stem. It has been shown that many small branches of the
vaguswithinthechestcan initiate LESrelaxation when stimulated directly (30).
Insummary,this study emphasizes several imlportanit
points conieerning the sympathetic iinnervation of the
LES. First, studies dturinig direct nieuiral stimulation mlay give results (uite differenit thani would be
pre-dicted from studies usinlg the exogenous
administra-tion of agonist or antagonist comnpound(Is. Second, the
LES does have an atropine-sensitive excitatory in-nervationthatiscarriedinthegreatersplanchnicnerve.
Third, the sympathetic innervatioin miay modulate the
action of direct vagal inihibitioIn on1 the LES. Fourth, although adrenergic inhibitionwithguanethidine aug-ments the excitatory, atropine-sensitive increase in
LESpressure during splanchnic inerve stimulation, the
true intrinsic role of the alpha anid beta adrenergic re-ceptors in the regulation of LES function remains unclear.
ACKNOWVLE DGMENTS
The authors wish to thank Ms. Marv Carroll for secretarial
assistance.
Thiswork wassupported by Research grant ROI AM 19379
from the National Institutes of Health.
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