normally evoked a gastric contraction, but caused relaxation in atropinized dogs.

With7text-figures Printed in Great Britain

GASTRIC VASODILATATION ANDVASOACTIVE INTESTINAL PEPTIDE OUTPUT INRESPONSE TO VAGAL STIMULATION INTHE DOG

BY S. ITO, A. OHGA AND T. OHTA

From the Department ofPharmacology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan

(Received 11 November 1987)

SUMMARY

1. Gastric vasodilatation, relaxation, and vasoactive intestinal peptide (VIP) output in response to vagal stimulation were studied in anaesthetized dogs.

2. Stimulation of the peripheral end of the vagus nerve (10Hz, 40V, 0.5ms) normally evoked a gastric contraction, but caused relaxation in atropinized dogs. Therewas nodetectable difference between the electrical thresholds for activation of the vagal preganglionic excitatory and inhibitory motor fibres.

3. Vagal stimulation also evoked gastric vasodilatation, which was blocked by hexamethonium but not by combined muscarinic and adrenergic blockade. Vagal fibres evoking vasodilatation had higher thresholds to electrical stimulation than those evoking motor responses.

4. Both gastric motor responses to vagal stimulation increased with increasing frequency up to 10Hz and a plateau between 10 and 40Hz, but the vasodilator response was significantly reduced above 20 Hz. Vagal stimulation at 10 Hz caused anincrease in gastric venousVIP output which was significantly reduced at40 Hz. 5. Low-intensity vagal stimulation (10Hz, 40 V, 0-05 ms) elicited gastric relaxation (40% ofa maximum), withno release of VIP orgastric vasodilatation.

6. It is concluded that release of VIP in response to stimulation of the

vagal

innervation to the stomach in the dog is primarily vasodilating in action.

INTRODUCTION

Stimulation of the peripheral end of the cut vagus nerve causes inhibition of gastric motilityinatropinizedanimals(Harper, Kidd&Scratcherd, 1959;Martinson &Muren, 1963; Ohga, Nakazato & Saito, 1969, 1970; Abrahamsson, 1973;Andrews & Scratcherd, 1980; Ohga, Nakazato & Ito, 1983; Ohta, Nakazato & Ohga, 1985). Vagal control of gastric motility is therefore considered to depend upon other transmitters in addition to acetylcholine.

Andrews & Lawes

(1985)

have

provided

evidence that there is no detectable difference in the electrical threshold for activation of

the

vagal excitatory and inhibitory fibres in the ferret.

The activation of

high-threshold

vagal

fibres has been reported to cause an increase in

gastric

blood flow in the cat

(Martinson,

1965).

Vagal

stimulation dilates the

gastric

submucosal arterioles of the rat

by

an atropine-resistant and

hexamethonium-sensitive mechanism

(Morishita

&

Guth,

1986).

VIP has been

implicated

as a neurotransmitter involved in the intestinal vasodilatation

which is elicited

by

mechanical

stimulation

of

the mucosa

(Fahrenkrug

et al.

1978b) and rectal vasodilatation

evoked

by

pelvic

nerve

stimulation

(Andersson,

Bloom, Edwards,

Jiirhult &

Mellander,

1983).

The

purpose

of

the

present

experiments

was to study in detail

the

gastric

motor

and vascular

responses

to stimulation of the

peripheral end ofthe cutthoracic

vagus

nerve

and their relation to

vagally

mediated

gastric venous VIP output in anaesthetized dogs.

METHODS Operative procedure

The

experiments

were

performed

on

thirty-three

adultdogsof either sexweighing between 8 and 13

kg

after an

overnight

fast.

Dogs

were anaesthetized with

chloralose

(50mg

kg-',

i.v.) and urethane

(100

mg

kg-',

i.v.)

after induction with

pentobarbitone

(30mg

kg-1,

i.v.). A tracheal cannula was inserted and the animals were maintained by intermittent positive-pressure ventilation.

Polyethylene

cannulae were inserted into thecephalicveinforsystemicadministration of

drugs

and into the

right

femoral

artery

for

recording

systemic blood pressure. The abdominal

cavity

was

opened by

incision of the left flank. The

spleen

andgreateromentumwere removed. For close intra-arterial infusion of

drugs

to the stomach, a polyethylene cannula was inserted

retrogradely

into a branch ofthe

splenic artery,

the

tip

ofwhich was placed at the origin ofthe branch. A 0.1ml bolus of

drugs

dissolved in sodium

phosphate-buffered

saline (PBS: sodium

phosphate,

10

mm; NaCl,

150

mM;

pH

7-4)

was

injected

through

the cannula andthen flushed out with0 15 ml of PBS. The

gastroepiploic

vein

running

along

the stomach was ligatedat the gastric antrum 2 or3 cm distantfromthe

pylorus

to

prevent

venous flow from the pyloricregion. Lateral

thoracotomy

was

performed

by removing

the 8th to

11th

ribs; hence the need for intermittent

positive-pressure

ventilation. A

communicating

branch of the leftvagusnerveto the dorsal vagus trunk inthe thorax was dissected free and its

peripheral

end mounted insilverring electrodes for

peripheral

vagal

stimulation. The

electrodes,

covered

by

a silicon tube, were insulated from the

surrounding

tissues

by

a

piece

of

gauze containing

liquid

paraffin. Square-wave pulses were

delivered from an electronic stimulator

(Nihon-Kohden,

SEN-3101). After heparinization (1000 U

kg-',

i.v.),

the

splenic

vein,

draining

the

gastric

corpus,

was cannulated and thevenousblood was

drained

continuously

into a siliconized beaker

through

a

photo-electric

drop-counter. Gastric

venous blood was collected

through

this cannula to measure plasma VIP, and venous outflow

pressure

was set at about 10

cmH2O.

The rectal

temperature

was maintained at 37

°C

with a heated table. After

warming

at about 37

C,

gastric

venous bloodwasreturned,via a cannulainserted into theexternal

jugular

vein,

by

meansof a

peristaltic pump

(Mitsumi

SJ1210).

Whenthe blood was

collected,

a

corresponding

amount of PBS was infusedfrom thejugularvein. In experiments for VIP

assay,

a branch of the left femoral

artery

was cannulated to collect arterial blood. In some

experiments,

both

splanchnic

nerves were cut between the 13th rib and thediaphragm.

Recordingprocedures

Arterial blood

pressure

was monitored

continuously

fromtherightfemoral arteryby a pressure

transducer

(Gold

Stantham

Inc.,

P23

IC)

throughout

the experiment. Gastric blood flow was

recorded

continuously

by

a

photo-electric drop-counter,

the electrical signals of which were

wasconnectedby a tube to a water reservoir placed on a displacement transducer, and the system wasfilled with warm water. The change in the weight of the water reservoir was recorded by the transducer (Ohga et al. 1970). The intra-balloon pressure was usually set at about 10cmH2O and the experiments were started 30min after the preparatory surgery. Vagal stimulation was applied at least 20 min after splanchnicotomy or treatment with blocking agents. In order to determine whether the recorded blood flow was restricted to the gastric corpus, 1 ml of Pontamine Sky Blue 6B (20 mgml-') was injected into thesplenic artery at the end of the experiment. After the dye appeared in the venous effluent, animals were killed by a rapid injection of pentobarbitone. The weights of the whole stomach and the dyed part of the stomach were measured. The mean weights ofthe stomach and the dyed gastric corpus region were 1097+3-4g (n = 13) and 26-9+1-7 g (n = 13), respectively.

VIP radioimmunoassay procedure

Theconcentration of VIP in venous and arterial plasma was determined by radioimmunoassay. Vagal stimulation was applied for 2min under various conditions. In each animal, two or three tests were performed at about 30min intervals, and the sequence of the test was randomized. Haematocritwas measured in the venous blood to estimate the volume of plasma. The blood was collectedfor 1 min or 30 s in an ice-cooled plastic tube containing the protease inhibitor aprotinin (Sigma, 800 U)and EDTA (Dojin, 12-5,umol). After centrifugation at 4°C, the plasma(0-8 ml) was extracted with atwofold amount of absolute ethanol. After centrifugation at 10000 g for 10min at 4°C, the supernatant was dried under vacuum on that day. The samples were kept frozen at -20°C untilassayed. Each sample was reconstituted in 40mM-sodium phosphate buffer (pH 7 4) attheoriginal volume and then assayed in duplicate or triplicate. Over 90 % of VIP in plasma was extractedbythis procedure and data were not corrected for recovery. In some experiments, plasma extracts obtained during stimulation were freeze-dried and subjected to gel-filtration analysis on a Sephadex G50 SF column (1 x 90 cm) eluted with 0 5M-acetic acid. Each eluting fraction was analysed by VIP radioimmunoassay after freeze-drying.

VIP (ProteinInc., Osaka, 0-6 nmol) was iodinated by the chloramine T method using 1 mCi Na "5I (Amersham) for radioimmunoassay. The reaction products were applied to a column (1 x 10 cm) of CM Sephadex C-25 equilibrated with 50mM-ammonium bicarbonate (pH 7-9), and then eluted with a linear gradient of equal volume (30 ml) of 50 mm- and 250mM-ammonium bicarbonate at a flow rate of 0-3 ml min-'. The radioactive peak fraction that eluted at about 250mM-ammonium bicarbonate was pooled. The iodinated VIP was aliquoted and lyophilized under a nitrogen stream. The radioimmunoassay was carried out by the method described previously (Ito, Ohta, Nakazato, Ohga & Yanaihara, 1986). The data from standards (0-78-100fmol) were analysed by the logit method and the best line was calculated by unweighted least squares usingapersonalcomputer (NEC9801). The equation for this line was used to estimate the VIPcontent ofunknowns. The gastric output of VIP was calculated as the veno-arterial difference inplasmaVIPconcentration multiplied by the gastric plasma flow, which was determined from a knowledgeofblood flow and haematocrit. The antiserum used(VK208) recognized the middle and carboxyl-terminal region of a VIP molecule and did not cross-react with the peptide histidine isoleucine. The intra- (n = 5) and inter-assay (n=5) coefficients of variation were +8-2 and +5-5% at thelower level of the standard (1-56 fmol) and + 13-4 and + 14-3% at the highest level of the standard (100fmol), respectively. The detection limit was 0-8 fmol.

Statistical analysis

Theresults were expressed as the mean+S.E.M. and statistical significance was assessed using Student's ttest (unpaired). P values less than 0-05 were considered to be significant.

RESULTS

Responses toperipheral

vagal

stimulation

General.Under steady-staterestingconditions,arterial blood pressure rangedfrom

Stimulationpulseduration (ms) 0 0-1 02 0-3 0*5

°r

-20k -40 I -601 100 r 50

F

QL 4 L 0 -I I 01 02 0-3 05 1

Stimulationpulse duration (ms)

Fig. 1. Stimulus pulse duration-response curves showing the vascular resistance and motorresponses in the stomach toperipheral vagal stimulation (10 Hz, 40 V) for 15 s in thepresence and absence of atropine. 0, the peak contraction (n =4) andvasodilatation (n=6) in the absence of atropine. 0, the peak relaxation (n=5) and vasodilatation (n=5) in the presence of atropine (1 mgkg-1). The motorresponses are expressed as a percentageof each motor response to vagal stimulation (U, 05ms).

0-20-5 1 2 5 ..*. 10 20 30 10 40 50 10 MABP 150 r (mmHg) 100 Bloodflow

I

(dropsmin-') 100~-Motorresponse (40mlH20)

Fig. 2. Effectsonnmeanarterial bloodpressure(MABP), blood flowandmotorresponses

in the stomach to peripheral vagal stimulation (0-5ms, 40V) for 15s at various frequenciesinthepresenceofatropine(1mgkg-').Numbers abovethetimescale(1min)

representstimulus frequencies (Hz).

a) cm -cU a) C.) 3 Cu Cu .U) Cu -O n a) C 0 0. 6a) 0 0 Stimulation (Hz) --L . ...~, ,,r rI .r r

I-1.-- 0AA A v- -A- %I'. 11AA .W"Yow

---- --I

0 5 ms)for 15 s either hadno effect onblood pressure (n = 10)orcaused a transient and small decrease in blood pressure of 8-5+0-5mmHg (n= 10), which was unaffected by atropine (1 mgkg-', i.v.). Vagal stimulation elicited an increase in gastric blood flow and a contraction, whichwasusuallyfollowed byarelaxation. In some preparations, the relaxation was obscured bylow gastric tone. The

latency

of these responses to vagal stimulation was 4-2±A1 s (n = 8) for changes in gastric blood flow and 37+02 s (n = 8) for gastric contraction.

Stimulationfrequency(Hz) 0 2 5 10 20 40

c,

0

2 -20 c-40-> -80 100 0 50 -° 20 (z 0 2 5 10 20 40 Stimulationfrequency(Hz)

Fig. 3. Frequency-response curves showing the gastric vascular resistance and motor responses to peripheral vagal stimulation (40 V, 0 5ms) at various frequencies for 15sin the presence and absence of atropine (1 mg kg-'). *, the peak contraction (n=4) and vasodilatation (n= 7) in the absence of atropine. 0, the peak relaxation (n= 7) and vasodilatation (n=7)in the presence of atropine. The motor responses are expressed as apercentage of each motor response to vagal stimulation (U, 10Hz).

In atropinized dogs (1 mg

kg-',

i.v.), the gastric contraction induced

by

vagal stimulation was reversedto

relaxation,

the

latency

ofwhich was 5-6+0.1 s (n= 9).

The vagally evoked relaxation and increase in blood flow were not affected

by

phentolamine (10 mg

kg-',

i.v.) or propranolol (2 mg

kg-',

I.v.) in combination (n =4). Atropine

significantly

prolonged the latency of the increase in blood flow (P< 005; 10-3±0-2 s, n= 9). All the responses to

vagal

stimulation were inhibited by hexamethonium (10mg

kg-',

i.v.).

Relative

threshold.

Vagal stimulation at 40V (10

Hz,

various

pulse

durations)

A B C +20 0

c~

m - -20 -60 150 c 0 *. 100 (U

+j~ ~~I

O 0. o 50 L

J.,

0-> 10 10 40 3i 0 400 c 300 E E 200 100 0 0 10 10 40 3mi Stimulationfrequency(Hz)

Fig. 4.Mean gastric vascular resistance, plasma VIP concentration, and VIP output in response toperipheralvagalstimulation (40 V, 0-5ms)for 2min( ) at 10 Hz (A, n=

5; B,n=8)and40Hz (C,n=6) inthe presence (B and C) and absence (A) of atropine (1mgkg-'). Symbols of the middle graph indicate arterial (0) andgastric venous (@)

VIP concentrations.

Frequency dependence. Figure 2 shows a typical continuous record of responses to vagal stimulation (40 V, 05 ms) at various frequencies for 15 s after muscarinic blockade. The increase in blood flow in response to vagal stimulation attained a maximum at 10 Hz. The increase in blood flow was substantially reduced by stimulation above 10 Hz but still discernibleduring stimulation at 50 Hz. The extent of the relaxation was not, however, reduced bystimulation at high frequencies.

Figure 3 summarizes the results of stimulation for 15 s at different frequencies before and after treatment with atropine (1 mgkg-', i.v.). Stimulation of the vagus nerve with a single pulse elicited cholinergic contractions, and the inhibitory motor response began to appear from 0 5 Hz. The excitatory and inhibitory motor responsesincreased with increasing frequency up to 10 Hz and a plateau between 10 and40 Hz. The vasodilator response to vagal stimulation started to appear at 0-5 Hz and attained a maximum at 10 Hz. Further increase in stimulus frequency caused a marked reduction in vasodilatation regardless of the presence or absence of atropine.

This reduction in the vasodilator response to vagal stimulation at higher frequencies might have been due to vasoconstriction. However, the vascular response to vagal stimulation at 40Hz was not modified by pre-treatment with phentolamine (10 mgkg-1,I.V., n= 3) or by splanchnicotomy (n=4). A combination of vagal stimulation at 40 Hz and a close intra-arterial infusion of isoprenaline (100 pmol) or VIP (10 pmol) resulted in a vasodilator response that was equal to the sum of the responses to each stimulation separately (n= 3). Neither the frequency-response curves for vasodilatation nor relaxation changed shape after treatment with neostigmine (0-2 mgkg-', i.v.), which was given to facilitate ganglionic transmission in four atropinized dogs, although both the relaxant and the vasodilator responses were potentiated at all frequencies tested. The reduction of gastric vasodilatation, but not relaxation, occurred to just the same extent in response to vagal stimulation at 40 Hz.

VIP output evoked by

vagal

stimulation

General. The vagus nerve was stimulated for 2 min and gastric venous blood was collected continuously before and after the start of stimulation. Gel-permeation column fractionation of theplasma extracts during vagal stimulation demonstrated onepeakof VIPimmunoreactivity whichelutedinthepositionexpected forporcine synthetic VIP. The gastric venous and arterial VIP concentrations were not

statistically different before and aftertreatmentwithatropine (1 mg

kg-',

i.v.). The restingVIP concentration in thegastric venousplasmawasslightly higherthan that in the arterial plasma regardless of the presence orabsence ofatropine in the same animal.

Frequencyofstimulation. As illustrated inFig. 4A, vagalstimulation(40V,0 5ms, 10 Hz) for 2min produced a large reduction of gastric vascular resistance accompanied byasubstantial risein venousplasmaVIP concentration without any change in arterial plasmaVIP concentration. Gastricvenous VIP output startedto increase within the first 30 s of the 2min period of stimulation and attained a maximum at the end of stimulation. The output of VIP then subsided

gradually

towards the control level. The time taken for VIP output to return to the control

A B C 0 oE .3x -50

\

ffl

-200 .A 03 I-ce -400 -60 .c 300 E 200-0. 0L 0-05 0-08 0 5 3 min

Stimulationpulseduration (ms)

Fig. 5.Meangastricrelaxation, vascularresistanceandvenousVIPoutputinresponseto peripheral vagal stimulation (40V, 10Hz)for2min()atvariouspulsedurations (A,

0 05ms,n=5;B, 0-08 ms, n=5;C, 0 5ms,n=8)inatropinized dogs (1 mgkg-'). The gastric relaxant responsesareexpressedas apercentageof the maximalrelaxation.C,the middle and lowergraphsweretransferred from the upper and lowergraphsofFig. 4B.

A B C X0_40 -c U -10 c -20 30 61 40 1000 E E . 500 0 0~ 4 2 min

Fig. 6. Mean gastric vascular resistance and venous VIP output in responseto intra-arterial injection of VIPat dosesof1pmol (A,n=6),2pmol (B,n=4) and4pmol (C, n=4) inatropinizeddogs (1 mgkg-'). Arrows indicate thetimeof infusion.

Stimulation (40V, 0-5ms) at40 Hz caused a smaller vasodilatation than that at 10 Hz (P< 0-05) (Fig. 4C),

although

both

frequencies

evoked very similar

gastric

relaxant responses. Stimulation at 40 Hz

produced

a small but

convincing

increase inthe gastric venous VIP concentration and in the output ofVIP

(P

< 0-05,n = 6). Thenet increase in VIP output for 7 min in responseto vagal stimulation at 40 Hz (131+21 fmol, n = 6) was

significantly

less than that evoked

by

stimulation at

10 Hz (P < 0-01).

The reduction of VIPoutput fromthe stomachinresponseto

vagal

stimulationat

40Hz was not affected

by

splanchnicotomy

(mean,

183

fmol,

n =

2).

Furthermore,

even when isoprenaline was infused

intra-arterially

to evoke vasodilatation

during

the period of stimulation at 40Hz, it failed to

modify

gastric venous VIP output (mean, 217fmol, n= 2).

more pronounced gastric relaxation than had stimulation for 15 s, because the relaxation increased gradually during sustained stimulation. Vagal stimulation with apulse duration of 0 05 ms elicited a gastricrelaxation, the magnitude of which was about 40 % of the maximal relaxation evoked by stimulation at 0-5 ms duration, but itproduced neither gastric vasodilatation nor an increase in gastric VIP output (Fig.

4 3 0 E 0. 0 CL 40 3 0 C 0 E cJ 0 a. C 2 2 1 A A A 1 2 3 4 5 0 5 10 15 20

Incrementof bloodflow(ml 3 min-')

Fig. 7. Relation between theincrementof gastric blood flow for3 minandVIPappearing inthe venous plasmainresponsetoVIPinjectionfor 3min(A) and toperipheral vagal stimulation at10Hzfor7min(B). A,thedosesofVIP(0,1pmol; *,2pmol;A,4pmol; * 10pmol). B, thepresence (El)and absence (A) ofatropine.

5A). When thepulsedurationwasincreasedto0-08ms, the

magnitude

of relaxation was increased to 65% of the maximum, and increases in gastric VIP output and vasodilatation were detected

(Fig.

5B).

VIP infusions

VIP (1-4pmol, I.A.)was

injected

afterpre-treatment with

atropine,

and

changes

inthe gastric vascular resistance and gastric VIP output were determined

(Fig.

6). VIP hadnoeffectonbloodpressureatthese doses. VIP(1 and 2

pmol)

hadnoeffect on gastric motility, but it caused a small

gastric

vasodilatation in all

preparations

response to exogenous VIPhad wornoff withinthe first 1 min collectionperiod after each injection. The gastric VIP output attained a maximum during the first collection period of injection and returned to the resting level over the next 3-4 min. The amount of exogenous VIP recovered inthevenouseffluent was 262+43fmol for 1 pmol (n= 6), 548+ 85fmol for 2 pmol (n = 4), and 1170+ 84 fmol for 4 pmol (n = 4), indicating that the mean recovery rate of exogenous VIP is 28-5 + 0-02 %. Correlation between vascular response and exogenous and endogenous VIP

The amount ofVIP appearing in the venous plasma for 3 min after intra-aterial injection was plotted against the subsequent increase in blood flow for 3 min (Fig. 7A), the values of which were transferred from the data shown in Fig. 6. A good positive correlation was obtained between them (n = 16, r = 0-87, P < 0-05, Y=

088X-02). The relation between the incremental changes in gastric venous VIP output for 7 min and blood flow for 3 minin response tovagalstimulation at 10 Hz is shown in Fig. 7B. The values were obtained from the experiments shown in Fig. 4Aand B. Positive correlations were observed before (n = 5, r= 0-56, P < 0 05, Y=

0-06X+0-12) and after (n= 8, r =0-92, P < 005, Y = 0 12X+0-44) treatment with atropine. Atropine made the regression line steeper. After muscarinicblockade, the slope of the regression line for exogenous VIP was about 7 times larger than that for endogenous VIP, indicating that VIP released by stimulation was much more effective ineliciting the blood flow response than exogenous VIP.

DISCUSSION

The present experiments confirmed the observation that stimulation of the peripheral end of the vagus nerve produces cholinergic contraction and NANC relaxation in the dog stomach (Ohga et al. 1970). In addition to the gastric motor responses, vagal stimulation also elicited a significant increase in gastric bloodflow. Thevascularresponse tovagalstimulation cannot beexplained bymetabolicfactors produced as aresult ofaugmented acid secretion, because acid secretion isblocked by atropine (Jansson, Lundgren&Martinson, 1970). Althoughthe increase ingastric blood flow induced by vagal stimulation seemed to be due, in part, to mechanical facilitationbygastric contractions and/or cholinergic vasodilatation,the increase in blood flow after muscarinic blockade was resistant to a- and

f8-adrenergic

blockade but wascompletely blocked by hexamethonium. It hasrecently been reported that vagalstimulation dilates the gastric submucosal arterioles of the rat via an atropine-resistantmechanism (Morishita &Guth, 1986). Furthermore, vagal stimulation also increases pancreatic (Hickson,. 1970) and portal blood flow (Fahrenkrug, Galbo, Holst &SchaffalitzkydeMuckadell, 1978a) in thepig after treatment withatropine. These results suggest that stimulation ofpreganglionic vagal nervefibres can cause gastric vasodilatation by activating postganglionic NANC neurones.

hypothesis is based on evidence that release of VIP, like gastric relaxation (Martinson & Muren, 1963), is only elicited by stimulation ofhigh-threshold vagal preganglionic fibres. In the present experiments, however, there was no detectable difference between the electrical stimulation threshold of the vagal preganglionic fibres driving the intramural NANC inhibitory neurones and those activating intramural cholinergic neurones in the dog, as was also the case in the ferret (Andrews & Lawes, 1985). Gastric vasodilatation and VIP output were evoked by activation of

higher-threshold

vagal

preganglionic

fibres than the

gastric

motor responses. In addition, both gastric vasodilatation andVIPoutput, butnot gastric relaxation, were reduced by increasing the

frequency

of stimulation to 40Hz. A similar reductionof the vasodilatorresponsehas been

reported

with

peripheral vagal

stimulation athigherfrequenciesinthecatpancreas (Greenwell &

Scratcherd,

1974). Moreover, theincrement of gastric VIP output in responseto

vagal

stimulationwas well correlated with that of the gastric bloodflow. Theseresultssuggest thatVIP is a neurotransmitter which is released from

postganglionic

neurones

innervating

the gastric vascularsmooth muscle, and suggest that the vasodilatornerves are distinct from intramural NANC motor inhibitory nerves in the

dog

stomach.

Gastric vasoconstriction isnot involved in the reduction of

gastric

vasodilatation and VIP outputoccurringwith vagal stimulation at

higher frequencies,

because the reduction of bothresponseswas notaffectedbypre-treatmentwith

phentolamine

or

by splanchnicotomy. Furthermore, stimulation at 40Hz did not increase systemic blood pressure, nor did it affect the vasodilatation evoked

by

VIP or

isoprenaline.

The reductionofvasodilatationand VIP outputoccurringathigher frequenciesmay be due to failure ofganglionic transmission. If

only

neuroeffector transmission had been depressed bystimulationat higher

frequencies,

ananticholinesterase would be expected to shift the

frequency-response

curve to the left to facilitate

cholinergic

ganglionic transmission. However, thiswas notthe casein the present

experiments.

VIP-containing neurones in themyenteric

plexus

andsubmucous

plexus

have been shown to

supply

terminals to circular smooth muscles and submucous arterioles in theguinea-pigsmallintestine,respectively

(Costa

&

Furness,

1983).Mostof the VIP-containing neurones in the submucous

plexus

have been

reported

to exhibit inhibitorysynapticpotentials

following

ashort-livedexcitatory

potential

inresponse to transmural stimulation of three

pulses

at 30 Hz

(Bornstein,

Costa &

Furness,

1986). Taking all these results together, vagal stimulation at 40 Hz may evoke inhibitory synaptic potentials in the VIP-containing neurones in the submucous plexuswiththeresult thatthey became less effective whenstimulated atsucha

high

frequency.

It has been

reported

that VIP

produces

either

endothelium-dependent (Davies

& Williams, 1984; Ignarro, Byrns, Buga &

Wood,

1987) or

endothelium-independent

arterial relaxation (D'Orleans-Juste, Dion, Mizrahi &

Regoli,

1985; Schoeffter & Stoclet, 1985). Itseems

likely

that the mechanisms ofVIP-mediated vasodilatation differ with respect tothe vessel type and species. The present experiments

provide

AND VIP OUTPUT

The present experiments do not negate the possibility that vagally mediated gastric relaxation is due to release of VIP. Vagal stimulation at 40 Hz elicited a maximal gastric relaxation but only a small amount of VIP was released into the gastricvenouseffluent.This might, however, have been sufficient to cause the gastric relaxation. Inanaccompanyingpaper (Ito, Ohga & Ohta, 1988), we report on further experiments carried out to elucidate the role of VIP in relation to gastric relaxation induced by NANC intramural inhibitory nerve stimulation.

We wish to thank Dr Y. Nakazata for his advice and reading the manuscript. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture ofJapan.

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