Vagal afferent pathway mediates physiological
action of cholecystokinin on pancreatic enzyme
secretion.
Y Li, C Owyang
J Clin Invest. 1993;92(1):418-424. https://doi.org/10.1172/JCI116583.
To establish the mechanism(s) and site(s) of action of cholecystokinin (CCK) on pancreatic secretion under physiological conditions, we used an in vivo model using anesthetized rats with pancreaticobiliary cannulas. Infusion of CCK-8 (10-160 pmol/kg per h) produced a dose-dependent increase in plasma CCK levels. CCK-8 infusion at 40 pmol/kg per h produced a plasma CCK level of 7.9 +/- 1.5 pM and an 80% increase in pancreatic protein output over basal. This level was closely approximated by a postprandial peak plasma CCK level by 6.2 +/- 1.1 pM. Pretreatment with atropine or hexamethonium completely abolished pancreatic protein response to low doses of CCK-8 (10-40 pmol/kg per h) but had only partial effect on doses > 40 pmol/kg per h. Bilateral vagotomy also abolished the pancreatic responses to low doses of CCK-8. Similarly perivagal treatment with a sensory neurotoxin, capsaicin, caused a complete inhibition of pancreatic protein secretion in response to CCK-8 infusion. In contrast, pancreatic protein responses to bethanechol were similar in control and capsaicin-treated rats. In separate studies we demonstrated that gastroduodenal but not jejunal application of capsaicin for 30 min abolished pancreatic protein secretion in
response to physiological doses of CCK-8. In conclusion, CCK at physiological levels stimulates pancreatic enzyme secretion via a capsaicin-sensitive afferent vagal pathway originating from the gastroduodenal mucosa.
Research Article
Find the latest version:
Vagal Afferent Pathway Mediates Physiological
Action of Cholecystokinin
on
Pancreatic Enzyme Secretion
Ying Li andChung Owyang
The University ofMichigan Medical Center, Gastroenterology ResearchUnit, DepartmentofInternal Medicine,AnnArbor, Michigan 48109
Abstract
To establish the mechanism(s)andsite(s) ofaction of
chole-cystokinin (CCK)onpancreatic secretion under physiological conditions, weused an invivo model using anesthetized rats
with pancreaticobiliarycannulas. Infusion ofCCK-8 (10-160
pmol/kgperh) produceda dose-dependentincreasein plasma
CCK levels.CCK-8 infusionat40 pmol/kgperh produceda
plasmaCCK level of7.9±1.5pMandan80% increase in
pancre-aticproteinoutput overbasal.Thislevel was closely approxi-mated by a postprandial peak plasmaCCK level by 6.2±1.1
pM. Pretreatment with atropineor hexamethonium completely
abolished pancreaticproteinresponse tolowdosesofCCK-8
(1040 pmol/kgperh) but had onlypartial effecton doses >40 pmol/kg per h. Bilateral vagotomy also abolished the pancreaticresponses tolow dosesof CCK-8.Similarly periva-gal treatmentwitha sensory neurotoxin, capsaicin, caused a
complete inhibitionofpancreatic proteinsecretion inresponse toCCK-8infusion.Incontrast,pancreatic proteinresponses to
bethanecholweresimilar in control andcapsaicin-treatedrats. In separatestudies we demonstrated that gastroduodenalbut notjejunalapplication of capsaicin for30minabolished
pancre-atic protein secretion in response to physiological doses of CCK-8.Inconclusion, CCKatphysiologicallevelsstimulates pancreaticenzymesecretion viaa capsaicin-sensitive afferent
vagal pathwayoriginating from the gastroduodenalmucosa.(J. Clin. Invest. 1993.92:418424.)Key words:
cholecystokinino
vagalafferentpathway*pancreaticsecretion*capsaicin*atro-pine*vagotomy
Introduction
Cholecystokinin (CCK) isthe
major
mediator ofpancreatic
exocrine secretion.Themechanism(s)
responsible
for CCK-in-ducedpancreaticenzymesecretionunderphysiological
condi-tions however is unclear. In vitrostudiesusing dispersed
pan-creatic acinidemonstrate that CCK-stimulated
amylase
release isinsensitiveto atropine ortetrodotoxin(TTX),'
indicating
directactionon
pancreatic
acini( 1).
Furthermore,
theeffects ofcholinergic agonists andCCK in isolatedpancreatic
aciniAddressreprint requeststo ChungOwyang, M.D., 3912 Taubman Center,Box0362,TheUniversityofMichiganMedicalCenter,Ann Arbor,MI48109.
Receivedfor publication 21 December1992.
1. Abbreviations usedinthispaper: 2DG,2-deoxy-D-glucose; TTX,
tetrodotoxin.
preparationsare no morethanadditive( 1, 2).Thissuggests the
effect of CCKonenzymesecretion ischolinergically
independ-ent(3, 4).However, invivostudies in humans (5-7) and dogs (8, 9) show thatpancreatic secretion stimulatedby CCKcanbe blocked by atropine, suggesting action via cholinergic
path-ways.Furthermore,enzyme outputin patients aftervagotomy
in response to low dosesof CCKis reducedcompared with normal controls (10). Itappearstherefore that CCKcan actvia both atropine-sensitive neural pathwaystostimulate
pancre-aticexocrinesecretion. Currently,it isnotknown ifexogenous
CCKinfusions that producephysiologicallevelsstimulate
pan-creatic secretionviacholinergic or noncholinergicpathways. Thisissuehasimportantphysiological ramifications. IfCCKat
physiological levelsstimulatespancreatic secretion viaan
atro-pine-sensitive pathway, then cholinergic neural pathways rather thanpancreaticacinar cellsrepresenttheprimarytargets onwhich CCKmay act as amajor mediator of postprandial pancreaticenzymesecretion.
Inthis study we investigated the mechanism and site of
action ofCCKon pancreaticenzymesecretionunder physio-logicalconditionsusing an invivorat model. Wehypothesize
thatCCKatphysiologicallevelsactsviastimulationof
cholin-ergic pathwayswhereassupraphysiologicalCCK levelsact di-rectlyonpancreaticacini. Furthermore, sincerecentstudies in theratsdemonstrate that afferentfibers oftheabdominalvagus
mediate CCK-elicited satiety signals (11), we examined the
roleof vagalafferentpathways in the mediation of CCK's
ac-tiononpancreaticenzymesecretion. Chemical ablation
stud-ieswerealsoperformedtoidentify specificvagal branches that
areinvolved in thisfunction.
Methods
Materials
Thefollowingwerepurchasedfrom SigmaChemical Co.(St.Louis, MO) atropine sulfate, bethanechol chloride, 2-deoxy-D-glucose
(2DG),andcapsaicin. Octapeptideofcholecystokininwaspurchased fromPeninsula Laboratories Inc.(Belmont, CA).
Experimental
procedures
Animal preparation. Male Sprague-Dawley rats, weighingbetween 250 and 300 g,werefastedovernightandanesthetizedwithamixture of xylazine and ketamine (13 and 87 mg/kg body wt, respectively). Throughamidlineincision,apolyethylenecatheterwasinserted into thecommonbile-pancreatic ductattheampulla. Asecond catheter wasplacedinto the duodenum,slightlyabove thesphincterof Oddi for intestinalperfusionofpancreaticobiliary juice.Theabdominalwound wascoveredwithasaline gauge and the animalwasmaintainedat37°C withaheating pad.
Pancreatic secretionstudy.After 60 min ofstabilization, combined bile-pancreaticsecretionswerecollected every 15 min. The volumewas
measured andanaliquotwastaken anddilutedwith distilledwaterfor proteindetermination. The remainder of the undiluted bilepancreatic
juicewasreturnedtothe intestine via the duodenal cannuladuringthe J.Clin.Invest.
© The AmericanSocietyfor ClinicalInvestigation,Inc. 0021-9738/93/07/418/07 $2.00
nextcollection period.Pancreaticobiliary juice protein was measured spectrophotometrically usingthe assay method of Bradford( 12).Our previous study ( 13)confirmedthat the increase inproteinoutputin thebile pancreatic juiceafter CCK-8stimulationmainlyreflected pro-tein from thepancreatic source. Biliary juice protein did not increase with CCKstimulation.
CCK-8dose-response studies. Graded doses of CCK-8 (10-160 pmol/kg perh) wereinfused for 60 min. Sixrats weretested for each dose.Pancreaticobiliary juicewascollected every 15 min. Blood sam-ples for CCKmeasurement wereobtainedatthe endof each infusion by cardiac puncture. Plasma CCK levelsweremeasuredbyabioassay method previously reported ( 14). In separate studies, postprandial plasmaCCK response wasdetermined by obtaining blood samples fromratsfed5 mlof 18% casein. Caseinwasinstilled into the stomach with anorogastric tube and blood samples were obtained 15 min after casein administration.
Atropine, hexamethonium, andTTXinfusion studies. To deter-mine the roleofcholinergicpathways in the mediation of CCK's ac-tion, atropine( 100,g/kgperh)wasinfused 30 min before infusion of CCK-8 andwasadministeredthroughouttheremainder of the experi-ment.Pancreaticprotein outputs andplasmaCCK levelswere com-pared with those obtained withoutatropine.Similar studieswere per-formedwith hexamethonium treatment ( 15 mg/kg bolus plus 7.5 mg/ kg per h continuous infusion) to determine the role of presynaptic cholinergicneuronsin the mediation of CCK-stimulated pancreatic enzymesecretion.
To exerttotal neuralblockade,TTX,anonspecificblockerof neuro-nal axoneuro-naltransmission,wasadministeredbyregional intraarterial in-fusion into thepancreaticarterythroughtheabdominalaortawith the ligationof the renal arteries and temporary blockade of the iliac bifur-cation.Aloadingdoseof10pg/kgTTXwasgivenfollowedby2pg/kg per h of continuousinfusion throughoutthe experiment.Atracheal cannulawasinsertedto securefreeairwaysand therats wereartificially ventilated. Complete neural blockade was established by demonstrat-ingthatpancreaticsecretion in responseto2DG stimulationwas com-pletelyabolishedbut responsestobethanecholremainedunaffected.
Bilateralsubdiaphragmaticvagotomy.To demonstrate that physio-logicallevelsofplasmaCCKactviastimulationofthevagalpathways, acutebilateralsubdiaphragmaticvagotomywasperformed. Througha midline incision oftheabdominalwall,the stomachwascarefully ma-nipulatedtoexposetheesophagus. Thesubdiaphragmaticvagal trunks wereexposedhalfway between the diaphragm and thegastriccardia. Bothanterior and posterior trunks of the vagalnerves weretransected. Forcontrolexperiments, theabdominalvagalnerves wereexposed but not cut.CCK dose-response studieswereperformedasdescribed.To demonstrate completeness of vagotomy,pancreaticprotein secretion in responseto2DGwastested.2DG,anonmetabolicableglucose ana-logue, causeshypothalamia, glucopenia,andexcites the dorsal vagal nuclei. It hastraditionallybeen used as an agentfortesting complete-nessof vagotomy ( 15, 16).Adoseof 75 mg/kg i.v. was used.
Perivagalapplication ofcapsaicin. To investigate the role of vagal afferent pathway in the mediation of CCK's action, we examined the effects ofperivagalapplication ofcapsaicin.Afteranesthesiathe abdom-inal vagal trunks were exposed.Asmallpiece of gauze soaked in0.1 capsaicinsolution(0.1 mlperrat)wasleftonthe vagal trunksfor 30 min.Solutions ofvehicle alone were applied to control rats. CCK dose-responsestudies as described above were performed 5 d after surgery in thesetwogroupsof rats.
Vagalafferentrootlet section. To further demonstrate that vagal afferent pathway mediates CCK's action on pancreatic enzyme secre-tion,parallelstudies with vagal afferent rootlet section were performed. Inthe rats selective afferent or efferent rootlet section is possible be-causetheafferent rootlet enters the medulla dorsal to the site where efferent rootlet exists. The rats were placed in a head holder with the aid ofanoperatingmicroscopy and the basal aspect of the occipital bone wasexposedbypostauricular scalpincision. This revealed thevagus
nerveasitpenetratedthroughtheduramater andseparatedinto
affer-entandefferentgroupsof rootlets. Thevagalafferent group consists of
two tofour rootlets that reach the dorsal lateral surface of the medulla. Throughthe dorsal approach the afferent rootletswereexposeddirectly and transected under direct vision. In the control group,craniotomy wasperformed in sham operations but rootlet sections were not per-formed. 2 d after left vagal afferent rootlet section, aright cervical vagotomy was performed before CCK dose-response studies.
Gastroduodenal mucosalapplication ofcapsaicin.Toinvestigateif theCCK-sensitive afferentnerveendings originate from the gastroduo-denal mucosa,weexamined the effects of mucosal application of cap-saicin in the stomach and duodenum ( 17). Capcap-saicin was also applied tothejejunum to examine regional specificity. After laparotomy, cap-saicin (6 mg/ml) was topically applied to the gastroduodenalorjejunal mucosa for 30min. Saline application was used in control rats. CCK dose-responsestudieswereperformed 4 d after local capsaicin applica-tion. Rats were checked for normal eye wiping movement, which indicates that local mucosal treatment of capsaicin has nosystemic effect( 17).
Bethanechol andCCK interactionstudies.Toinvestigate ifthe stim-ulatory action of CCK requires the presence of a cholinergic tone,we examined the effect ofbethanechol,amuscarinic receptoragonist,on CCK-evoked pancreatic secretion in vagotomized rats. Acute subdia-phragmatic vagotomy was performed asdescribedabove in two groups of rats. In the first group of rats, which served as the control, aftera 60-min basalperiod,pancreatic protein secretion in response to intrave-nousinfusion of bethanechol(0.5mg/kg perh)wasmeasuredfor a total of 120min. The second group of rats received intravenous infu-sion ofCCK-8 (40pmol/kgperh)alone for 30 min followed by concur-rentintravenous administration of bethanechol (0.5mg/kgperh) for anadditional 120min.
Statisticalanalysis
Results were expressed as mean±SE. The multivariate analysis of vari-ancemethod was used to evaluate the effect of the repeated measure-ment overtime,treatmenteffect,and theinteractionbetween them. Significance was accepted at the 5% level.
Results
Plasma CCK levels
Aftera 12-h fast, the basal plasma CCK levels were 0.5±0.1 pM. The basal levelswere notaffected by vagotomy (0.6±0.1 pM)oratropine administration (0.55±0.09 pM). Intravenous infusion of CCK produced a dose-related increase in plasma CCK levels (Fig. 1). The plasma CCK levels during infusion of 40 and 160 pmol/kg per hwere7.9±0.5 and 19±2.5 pM, re-spectively. Plasma CCK responsestoCCK infusion were not affectedby bilateral vagotomy (Fig. 1 )oratropine
administra-tion(datanotshown).
Todetermine which rate ofCCK-8 infusion would produce aphysiological plasma CCK level, we measured plasmaCCK
levels after administration of a casein meal. Casein feeding re-sultedinapostprandial peak increase in plasma CCK levelsto 7.3±1.1 pM (Fig. 1 ). Therefore it appears that the infusion of CCK-8 at 40pmol/kg per h produced a plasma CCK level
(7.9±0.5 pM)similar tothatseenphysiologically afteracasein meal(7.3±1.1 pM). In contrast, the 80 and 160 pmol/kg perh doses produced markedly supraphysiological plasma CCK lev-els(Fig. 1).
Pancreaticsecretionstudies
The basal pancreatic protein secretion was stable, averaging 130±18 mg/h. Intravenous infusion ofCCK-8 produced a dose-dependent increase in pancreatic protein secretion (Fig.
2).
CCK-8 infusion of 40 pmol/kgperh, whichproducedaPlasma CCK Response
Exoaenous
CCK
Infusion
24
am4 U
UL
E
S a
24
20
O
Intact
-e
Vagotomy lb
12
8
4
0
0 20 40 80 160
CCK-8
(pmollkg/h)
IV
Infusion
level, causedan80% increase in pancreatic proteinoutput over basal(Fig. 2). On the other hand, infusion of supraphysiologi-cal doses of CCK-8 at 80 and 160pmol/kgper hproducedan
180 and 238%increase in pancreatic proteinoutput over basal
(Fig.
2).
Effect
ofatropine and hexamethonium. Administrationof atropine (100,g/kg
perh)orhexamethonium (15 mg/kgfol-lowed by 7.5 mg/kg per hr) did not affect basal pancreatic
output butcompletely abolished pancreaticresponses to
CCK-8infusionat10, 20, and 40 pmol/kgper h(Fig. 2).In contrast,
atropineorhexamethonium produced only partialor no
inhibi-o 250 0 2 0 = 200
. E
0- 150
0-n. a
a
sX 100
a-.0
h. 50
A* a
l Control a Atropine
El Hexamethonium
rn**T
L 1 1
I
10 20 40 CCK-8 (pmol/kg/h)
**
80 160
Figure 2. CCK-8 infusion evokedadose-dependentincrease in pan-creaticproteinsecretion. Doses>40pmol/kgperhareconsidered supraphysiological. Administration ofatropine( 100gg/kgperh)and hexamethonium ( 15mg/kg+7.5mg/kgperh) completelyabolished pancreaticresponsestophysiologicaldosesof CCK-8(c 40pmol/kg
perh) butweremuchless potentathigherCCK-8 doses(80-160
pmol/kg perh). Valuesaremean±SE for sixratsin each group. *P <0.05.
i
()
U
E
S00 S
Fasting
Casein
Figure 1. Infusion of chole-cystokinin octapeptide (CCK-8) induced dose-de-pendentincrease in plasma CCKlevels. Infusion of CCK-8 at 40pmol/kg per h produced plasma levels simi-lar to those seen after a casein meal. Vagotomy had no ef-fectonbasal and CCK-8 in-fusion-stimulatedplasma CCK levels. Values are mean±SE for six rats in each group.
tion ofpancreatic responses to supraphysiological doses of CCK-8(Fig. 2). Theseobservations indicate that at physiologi-cal levels, CCK acts at a presynaptic site of the cholinergic pathway, whereas at supraphysiological levels CCK acts
mainly viaanatropine-insensitive pathway.
Effect ofvagotomy.Immediatelyafter vagotomy there was nochangein basalpancreaticprotein secretion. However, the responses to physiological doses of CCK-8 (10, 20, and 40 pmol/kg per h) were completely abolished (Fig. 3). This sug-gests that the vagal pathwayis the primary site of action of CCK under physiological conditions. In contrast, vagotomy
C
0
250 0 Control
0~~0T
200
-0,
100
,0 5
50
0
10 20 40 80 160
CCK-8 (pmol/kg/h)
Figure 3. Immediatelyafter vagotomy (TV) therewas nochange in basalpancreatic proteinsecretion but the responsestophysiological doses (< 40pmol/kg/hr) of CCK-8werecompletely abolished.In contrastvagotomy hadnoeffectonresponsestosupraphysiological levels of CCK-8. Infusionof tetrodotoxin (TTX) (10ag/kg+2,g/
hadlittleor noeffectonresponses tosupraphysiological levels
ofCCK-8 (80 and 160pmol/kgperh) (Fig. 3). To
demon-stratethatwehaveperformed completevagotomy westudied the effect of2DG, which has previously been shownto
stimu-latepancreatic secretion viaavagal cholinergic pathway ( 15, 16). In intact rats, 2DG (75 mg/kg) caused an increase of
pancreatic proteinsecretion by 150% overbasal. In ratswith truncalvagotomy,therewas noincrease inpancreatic secretion inresponse to2DGadministration.
Toinvestigatethe mechanismsresponsible for pancreatic
response tosupraphysiologicallevelsofCCK,weexamined the effect of intraarterial infusion of TTX, which exerts total neural blockade. TTX inhibited 65-75%ofthepancreatic re-sponses toCCK-8infusionat80 and 160pmol/kgperh(Fig.
3).Inseparatestudies,wedemonstrated thatintraarterial ad-ministration of TTX completely abolished pancreatic re-sponses to2DG but had no effecton stimulation evoked by bethanechol, which acted directlyonpancreatic acini. These
observationsindicate thatsupraphysiologicallevelsofCCKact onintrapancreaticneuronsanddirectlyonpancreaticacinito
mediate pancreaticenzymesecretion.
Effectofperivagal application ofcapsaicin. Similar to trun-cal vagotomy, perivagal application ofcapsaicin completely abolishedpancreatic proteinsecretion inresponseto
physiolog-icaldosesofCCK-8 infusion (10, 20, and 40 pmol/kgperh) buthadnoeffectontheresponses tosupraphysiologicaldoses (80 and 160pmol/kgperh)(Fig. 4).In contrast, we demon-strated. thatpancreatic protein secretion inresponse to 2DG stimulation remained intact in rats with perivagal capsaicin
treatment,indicatingthatcapsaicindidnotaffectefferent
va-gal function(Fig.5).
Effect
ofvagalafferent
rootletsection. Tofurtherdemon-stratethatvagalafferent pathway mediates CCK's action on
pancreaticenzymesecretion, parallelstudies with vagal
affer-entrootletsectionwereperformed.Similartoperivagal
appli-cation ofcapsaicin, afferent rootlet section also completely abolishedpancreaticresponses tophysiologicaldosesof CCK-8(10, 20,and 40pmol/kgperh)butwasineffectivetomodify
300
C
o 0 Control 0 Capsaicin
Cf E [3 Afferentrootlet section
C
Co
200
0
0-*O100
co0
C* *
10 20 40 80 160
CCK-8 (pmol/kg/h)
Figure4.Perivagal application of capsaicin or vagal afferent rootlet section completely abolished the pancreatic responses to physiological doses(< 40 pmol/kg per h) of CCK-8 but had no effect on the re-sponsestosupraphysiological doses. Values are mean±SE for six rats ineachgroup. *P < 0.05.
E
c
0 6
I-0
0
Vehicle
Capsaicin
0 30 60 90 120
Time (min)
Figure 5. Pancreatic protein responsesto2DGstimulation remained intactinratswith perivagalcapsaicintreatment,indicatingthat cap-saicindoes notaffectefferentvagalfunction.Values are mean±SE for sixratsin each group.
theresponses to supraphysiological doses of CCK-8 (80 and 160pmol/kgperh)(Fig.4).
Effect
ofgastroduodenal mucosal application ofcapsaicin.Compared with control, gastroduodenal mucosalapplication ofcapsaicincompletelyabolishedpancreatic proteinresponses tophysiologicaldosesofCCK-8 (10,20, and 40pmol/kgper
h) but had no effect on the responses to supraphysiological
doses(80 and 160 pmol/kgperh)(Fig.6).In separatestudies
wedemonstrated thatgastroduodenalmucosalapplication of
capsaicindidnotaffectpancreatic proteinresponses to stimula-tion by 2DG, indicating that gastroduodenaladministrationof
capsaicin didnotaffect efferentvagaltransmissionor
pancre-aticacini secretory function.
In contrast to duodenal application of capsaicin, jejunal mucosalapplication of capsaicindidnotaffectpancreatic
pro-tein responses to both physiological and supraphysiological
dosesofCCK-8(Fig.6).
300
C
O 3 Vehie
@ 250- E3
Jejuno-capsaian
c 200* UDuodeno-capsaicin200 C
so
150-*0
10000
A0 0
20 40 80 160
CCK-8 (pmol/kg/h)
Effect of bethanecholon CCK-evokedpancreatic secretion
in acutevagotomizedrats.Intravenous infusion of bethanechol
(0.5 mg/kgperh)inacutevagotomized rats produced a 65% increase in pancreatic protein secretion over basal. On the other hand, administration of CCK-8 (40 pmol/kg per h) alonefailedtostimulate pancreatic secretion in these rats(Fig. 7). Furthermore infusion of CCK-8 also did not evoke addi-tional increase inpancreatic secretion stimulated by bethane-chol (Fig. 7). This indicates that failure of CCK-8 given at
physiological
dosestoevoke pancreatic secretion in acutevagot-omizedratsisnotduetoabsence of cholinergic tone.
Discussion
Themechanism(s)bywhichCCK acts to stimulate pancreatic enzyme secretionremainscontroversial. In this study we
inves-tigated
the relative roles of neural activation and direct acinistimulation at physiological and supraphysiological levels of CCK
using
an in vivo rat model. Our results clearly indicate that the sitesofCCK'sactionto stimulate pancreatic secretionaredosedependent. DosesofCCK-8that produce physiologi-cal
plasma
CCK levels actvia stimulation of the vagal afferentpathway,
whereas doses thatproducesupraphysiological CCK levelsact tostimulate intrapancreatic neurons and to a lesserdegree pancreatic
acini. Theseobservations support a predomi-nantly neuralphysiological rolefor CCK to stimulate pancre-atic enzyme secretion.Inthe pastanumberofinvestigatorswereunable to
demon-strate inhibition ofCCKorCCK-like peptide-evoked pancre-atic enzymesecretionwithatropinein the dog ( 18, 19). These studies all used
high
dosesofexogenous CCK and did notas-sesswhether these CCK doses reproduced physiological
condi-tions. On the otherhand, Kontureketal. (20), using a canine
model, demonstrated that pancreatic enzyme outputs in re-sponsetolowdosesof exogenousCCKwasinhibited by
atro-pine,
whereas enzyme outputs in response to high doses of300
250
9
200-0
-
150-
100-Bethanecol infusion in both groups
I I
0 Control - CCK-8
CCK-8iuson
0 60 120
Time (min)
180 240
Figure7.Effect of bethanechol on CCK-8-evoked pancreaticprotein secretion inacutevagotomized rats. After acute subdiaphragmatic vagotomy, bethanechol (0.5 mg/kg per h) was administered by con-tinuousintravenous infusion for 120 min in both groups of rats. In onegroup, CCK-8 (40 pmol/kg per h) was infused 30 min before bethanechol infusion and continued for a total of 180 min. Values aremean±SEfor sixratsin each group. No significant differencewas observed between the two groups.
CCK were relatively insensitive to atropine. These observa-tionsare very much in accordance with our present findings.
Unfortunately, in that study, the CCK preparation used was
impure and there were no determinations of plasma CCK made intheir
investigation,
therefore, no assessment ofphysio-logical relevance could be made. More recently we (5) and others(6, 7) have shown that, in humans, CCK infusion that producedplasma CCK levels similar to those seen
postpran-diallystimulated pancreatic secretion by an atropine-sensitive pathway. Theseobservations suggest that in both humans and
experimental animalscholinergic neural pathways rather than pancreatic acini represent the primary targets in which CCK
acts to stimulatepancreatic enzyme secretion.
Sinceatropine andhexamethonium completely abolished
pancreaticenzyme responses to physiological doses of CCK, it suggests that CCK is acting on a presynaptic site along the
cholinergic pathway.Tofurther identify the location of action we examined the effect of bilateral subdiaphragmatic vagot-omy. Similartoatropine orhexamethonium, vagotomy com-pletely abolished pancreatic responses to low (physiological)
dosesof CCK but had little effect on responses to supraphysio-logical doses of CCK-8. We ruled out the possibility that a basal vagal cholinergic tone is necessary for CCK to directly
stimu-latepancreatic acinar secretion by showing that CCK did not produce an additional increase in pancreatic secretion
stimu-latedby bethanecholafter acute vagotomy (Fig. 7). Our results thereforeindicate that CCKstimulatespancreatic secretion via vagal pathway.This observation is supported by previous stud-iesshowingreductionin basal and nutrient-stimulated pancre-atic secretionafter truncal vagotomy (10, 18). Additionally, Malagelada et al. ( 10) reported that pancreatic secretion
in-duced by low but not high doses of CCK is impaired after
vagotomyin humans. Furthermore, studies on dogs with
dener-vated autotransplanted pancreas show impaired
responsive-ness to nutrients (21 ). Subsequent vagotomy or
administra-tionofatropinedoesnotfurtherimpair pancreatic responses to nutrient stimuli in this model (19, 21). These observatiqns indicatethe importanceofvagalinnervation in the mediation
ofCCK-evokedpancreaticenzyme secretion. Itisimportant,
however, to contrast these findings with some earlier studies
that show thatvagotomydoesnotsignificantlyreduce pancre-atic enzymesecretioninresponsetoexogenousCCK ( 18, 21-24). These contradictoryfindings are likely to be related to the
use ofsupraphysiological doses of CCK in earlier studies and differentlengths oftimeafter vagotomy before theexperiment
begins.In fact ourpreliminarystudiesinratsindicate that
re-sponsiveness of the pancreas to CCK stimulation returns
within2 wkafter vagotomy,possiblybecauseofrecruitment of
apopulation ofintrapancreaticneuronsthatwere
previously
unresponsivetophysiological concentrations of CCK (unpub-lished data).
To investigate ifCCKexerts itsaction viaanafferent or
efferent vagal pathway we examined the effect of
perivagal
treatmentwith thesensory neurotoxincapsaicin.
Capsaicin
is becoming widelyused as a tool toinvestigate the roleofaffer-entC-fibersin a number ofphysiologicalprocesses(25).
Sys-temic administrationofcapsaicinhas been showntoaffectneu-rotransmission inall somatic andvisceral capsaicin-sensitive
fibers. In addition, since capsaicin crosses the blood-brain barrier, systemic administrationofcapsaicin mayalso
produce
effects in the centralnervous system.Inthisstudywe
applied
affer-ent terminals in the peripheral and central nervous system. Perivagal application ofcapsaicin has been shown to inhibit axonaltransportofpeptides including substance P and
somato-statin (26). Previous studies have demonstrated thatperivagal
capsaicin treatment abolishes the vagal afferent pathway by
which CCKincreasessatiety(11)and decreasesgastric
empty-ing (27). Weshowed thatperivagalpretreatmentwith
capsai-cin impaired pancreatic responses to physiological doses of CCK-8, an effect similarto that observed with vagotomy or
atropine. Similar findings were observed with vagal afferent
rootletsection. This indicates thatvagalafferentpathwayis the primarysiteof actionofCCK. Toensurethatcapsaicin treat-ment does notaffectefferentvagal function,wedemonstrated that pancreaticresponseto2DG stimulation remained intact incapsaicin-treatedrats.Sincepancreaticresponsesto stimula-tion byphysiologicaldoses of CCKwereimpairedin these rats,
whoseefferent vagalfunctionwasintact,itarguesagainstCCK acting onvagalefferentpathwaytomediatepancreatic
secre-tion. We have previouslydemonstrated that CCK stimulates acetylcholine release from rat pancreatic lobules, indicating
CCKcanevokeintrapancreaticcholinergictransmission(5).
Howeverthe doses of CCK-8(1 ,uM)neededtostimulate
intra-pancreatic nervesin the lobule preparationwere > 106times
significantly higher than postprandial levels ofCCK, raising questionsas tothe
physiological
relevance of this observation. Ourcurrentin vivostudies further demonstrated thatonly
su-praphysiologicallevelsofCCKactonintrapancreaticneurons to mediatepancreaticenzymesecretion
(Fig. 3).
Using combined immunohistochemistry and retrograde tracing,thepresenceofcapsaicin-sensitiveafferentnervefibers
has been shown in the gastrointestinal mucosa (28). These neuralfibersareknowntoparticipateinthe
regulation
ofmotil-ity, acidsecretion, andbloodflow( 17,29).Tofurtherlocalize the site of action ofCCK on the vagal afferent
pathway
weexaminedtheeffectofmucosalapplicationof
capsaicin
indif-ferent regions ofthe
gastrointestinal
tract.Thistechnique
has been usedto chemically denervate sensory fibers in the duo-denalmucosa(17). Ourstudiesdemonstrated thatgastroduo-denalapplication ofcapsaicin completelyabolished
pancreatic
secretoryresponses to
physiological
dosesof CCK-8 but hadnoeffectontheresponses tosupraphysiologicaldoses.In contrast,
jejunal mucosalapplicationofcapsaicindidnotaffect
pancre-atic responses to CCK-8. These observations indicate that
CCK-sensitive afferentfibers originate fromthevagalbranch fromthe mucosaofthestomach and duodenum butnotfrom
thejejunum.
Inconclusion, wehave demonstrated that themechanisms
andsitesofactionofCCKonpancreaticenzymesecretionare
dose dependent. Doses of CCK-8 thatproduce physiological
plasmaCCK levelsactviastimulationofvagal afferent
path-way originated from thegastroduodenal mucosa(Fig. 8). In contrast, doses of CCK-8 that produce
supraphysiological
plasma CCKlevels act on intrapancreatic neuronsand, to a lesser degree, onpancreaticacini(Fig. 8).Thesefindingshave
important physiological ramifications. Not only do they
ex-plain thediscrepancies inprevious in vivoversusinvitro stud-iesbuttheyrevolutionizeour currentconcepton the mecha-nism of action ofCCKonpancreatic exocrine secretion. Under physiological conditions, cholinergic vagal afferent pathway
rather thanpancreatic acinarcells representtheprimarytargets on which CCKmay act as amajor mediator ofpostprandial
pancreaticenzymesecretion.
Figure 8. Thesitesand mechanisms of action of CCKtostimulate pancreaticenzyme se-cretion. Doses of CCK-8 thatproduce
physiologi-cal plasmaCCK levels actviastimulationof
vagal
afferentpathway CCIC_elK
lm._
originated
fromthegas-troduodenalmucosa.
dos)
eIncontrast, doses that
produce
supraphysiolog-Ch. rw nwrm ical plasma CCK levels
actonintrapancreatic
CCK neuronsand,toalarger
(Supraphso"okic
dos) extent,onpancreatic
acini.
Acknowledgments
Thisinvestigationwassupported in partby U.S. Public Health Service grantsDK-32838 and P30 DK-34933 from theNational Institute of Diabetes, Digestive,andKidneyDisease.
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