The enteric nervous system modulates mammalian duodenal mucosal bicarbonate secretion

1993;105:410-417

GASTROENTEROLOGY

The Enteric Nervous System Modulates Mucosal Bicarbonate Secretion DANIEL

L. HOGAN,

Division of Gastroenterology,

BIGUANG

YAO, JOSEPH

Mammalian

H. STEINBACH,

Duodenal

and JON I. ISENBERG

University of California at San Diego, San Diego, California

Back#ound: Interaction of the enteric nerves in regulating mammalian duodenal mucosal bicarbonate secretion Is not well understood. The purpose of the present experiments was to evaluate the role of the enterlc nervous system on bicarbonate secretion from rabbit duodenal mucosa in vitro. Methods: Proximal duodenum from male New Zealand White rabbits was stripped of seromuscular layers, mounted in Usslng chambers, and studied under short-circuited conditions. Effects of electrical field stimulation, vasoactive intestinal polypeptide (VIP), carbachol, prostaglandin E, (PGE,), dibutyryl-cyclic adenosine monophosphate (db-CAMP), and the neurotoxin tetrodotoxin (TTX) and muscarinic blockade by atropine were studied. Results: Electrical field stimulation significantly (P < 0.01) stimulated bicarbonate secretion, short-circuit current (lsc), and electrical potential difference (PD) that was sensitive to both TTX and atropine. VIP-stimulated bicarbonate secretion was significantly inhibited by TTX (-73%), yet Isc and PD remained unchanged. Atroplne decreased VIP-induced bicarbonate secretion (-69%) and Isc (-43%). Carbachol-stimulated bicarbonate secretion, Isc, and PD were abolished by atropine, whereas TTX was without affect. Neither TTX nor atropine had a significant effect on PGE, or db-CAMPstimulated bicarbonate secretion. Conclusions: These results suggest that (1) enteric nerve stimulation activates an acetylcholine receptor that in turn stimulates duodenal epithelial bicarbonate secretion; (2) VIP stimulates bicarbonate secretion, in large part, via the enteric nervous system; and (3) PGE, and CAMP stimulate bicarbonate secretion independent of the enteric nervous system.

separate lated

transport

pathways

bicarbonate

bene-2,2’-disulfonic whereas dent

acid

DIDSinduced

PGE,-stimulated tionable.9J0 carbonate

bicarbonate

transport

process

tory pathways that surround villi.“-‘3 nerve

terminals

and histamine)

active,

and neural

into the

in rat ileum

that

or choliner-

and acetylcholine,

candidates (e.g., VIP, isoleucine

to influence

stud-

nerve fibers

and extend

adrenergic

and secre-

Anatomic

of enteric

to noradrenaline of other

an

bicarbonate

shown

may be either

tion as neurotransmitters [NPY], peptide histidine statin,

nerves

crypts

it has been

are a number

is

both electrogenic

in regulating

a dense network

Also,

bi-

pathways.

of the enteric

gic. I4 In addition there

and

involving

transport

the duodenal

ques-

duodenum,

is not fully understood.

ies have shown

of VIP- and

Na+ and Na+, K+-adeno-

(ATPase)

mediators

one-

remains

in mammalian

requires

and electroneutral The interaction

by approximately

CAMP plays a role in stimuits mediation

transport

channel).

the rate of VIP-

secretion,

triphosphatase

nonneural

is indepen-

HCO;

reduced

secretion

Therefore,

energy-dependent

Cl--sensitive,

secretion

intracellular

bicarbonate

and

(i.e.,

solutions

bicarbonate

half. Whereas

sine

(DIDS)-

exchange

and Cl--free

PGE,-stimu-

is 4,4’-diisothiocyanostil-

db-CAMP-stimulated

of HCO;/CI-

lating

are involved.

secretion

that may funcneuropeptide Y [PHI], somato-

bicarbonate

trans-

port. In secretory

states

with

preserved

intact

intestinal

epithelium, it has been proposed that reflex activation occurs via stimulation of a receptor that releases peptides and/or amines into the interstitial space, thereby in turn meactivating adjacent nerves. I5 Interneurones

he proximal duodenal epithelium transports biinto an adherent mucus layer, thereby carbonate T providing a protective barrier from damage caused by reported, in acid and pepsin, ‘-* We have previously

diate the transmission of the nerve signal to the submucosal plexus and the efferent neurones via cho-

rabbit duodenum in vitro, that duodenal mucosa bicarbonate secretion is stimulated by vasoactive intestinal polypeptide (VIP), prostaglandin E, (PGE,), dibutyryl-cyclic adenosine monophosphate (db-CAMP), and theophylline and inhibited by anoxia, ouabain, and Na+-free solutions.“,” Furthermore, at least two

Abbreviations usedin this paper: db-CAMP, dibutyryl-cyclic adenosine monophosphate; DIDS, 4,4’-dlisothiocyanostllbene-2,2’-disulfork acid; EFS, electrical field stlmulation; I,, short-clrcult current; J,, net Ion flux; PHI, peptlde hlstldine isoleucine; R, electrlcal reslstance; TTX, tetrodotoxin. 0 1993 by the American Gastroenterological Association 0016-5085/93/$3.00

August

ENTERIC

1993

linergic,

nicotinic

transmitters

postganglionic

at the effector

tylcholine

and/or

Hubel16 short-circuit

ileum

(I,,),

field

potential

changes

difference

in

(PD),

Isc and I’D were not

yet were blocked

tetrodotoxin

(TTX).

have, in part, been confirmed

totally

These

by the

observations

and extended

additional species. 17-19The results enteric nerves have a significant

addition, HCO,-

, 25; HPOa2contained

Experiments Physiologic

linergic

and

atropine-resistant

nerve cho-

period

of the present

study was to evaluate

nervous

system

bicarbonate

secretion

and possible

interacting

the

on proximal

duo-

and to explore

the

roles of VIP and

receptors.

cuit electrical and

in

ute periods

averaged

cal resistance PD. After

Carbachol purchased

La Jolla,

and

CA) and the late Dr. Andre

Kalamazoo,

Co.,

db-CAMP,

MI),

indomethacin,

respectively.

and

TTX

were

were obtained

from

Fisher

Scientific

were

male New Zealand

White

prevent

prostaglandin

traperitoneally After

duodenum

serosal

and muscle aerated

mounted

(starting

a circular between

aperture

a few millimeters

solution

two Lucite

luminal

bathing

distal to the

The

mucosa

of

America,

Westlake,

OH).

W-P

pulse by a lo-microsecond at a frequency with a stimulus

W-P Instruments). occurred

evaluate

the optimum pilot

Peak HCO,-

quency

setting

was divided

(Table

1).

In subsequent utes followed reapplied

100-V positive 100-V stimuli

stimulus

measurements.

were

for this

performed

to

in 10 tissues: 5 Hz, 10 Hz,

frequencies

of either

5 10 or 10 10 micro-

and I,, responses

experiments,

830;

of EFS was

conditions

experiments

by a 30-minute

(model

potential

ion flux, no interference

of 10 Hz and duration

for a second

These

generator

electrical

to the transmucosal

the following

with

The rate of lu-

isolation CT). The

of 10 Hz for 600 milliseconds

Because

preparation,

of the tis-

Hamden,

interval.

interval

and 20 Hz with wave durations

(10 mL) was

surface

from the EFS in the other electrical

To ascertain

to

to a stimulus

from a 500-microsecond,

each second

the

the tissue parallel

wave form was a 500-microsecond, pulse separated

on

via a pair of aluminum

Instruments,

seconds.

was

circulated with a 100% 0, gas lift system. Luminal pH was maintained at 7.40 by continuous titration with an isotonic solution containing HCl (25 mmol/L), by pH-stat (ETS 822; Radiometer

through

on the submucosal

850A;

based

et a1.‘6~‘9~20In brief,

were connected

were applied

tissue

were measured

was

Hubel

propria

mucosa

experiments.

solution

was passed

in a nonbuf-

half-chambers

paired

of the

nerves

et al. and

placed

perpendicular

(3 mg/kg

and stripped

dissection

(37°C).

to provide

in-

intramuscularly

and a 5-cm segment

of 1.23 cm’. Each

two chambers

The unbuffered

(20 mg/kg

layers by sharp between

kg. To

(4 mg/kg

Isc,

Depending

At least four tissues were studied

of enteric

of the muscularis

negative

1 hour before surgery.

bile duct) was isolated

Ringer’s

vertically

2.5-3.0

IM), and acepromazine

was opened

entry of the common fered

weighing indomethacin

with ketamine (10 mg/kg

IM), the abdomen proximal

rabbits

synthesis,

on overnight-fasted,

[IP]) was administered

anesthesia

[IM]), xylazine

performed

Isc and

secretion,

series.

current

rectangular

Experiments

for 20 minutes.

electrical

stimulus

Study Design

The electri-

to stabilize

the plane

(model

indicated.

at

lo-min-

from the recorded bicarbonate

period.

bicar-

recorded

the tissues,

of Cooke

unit

for a brief

the open-cir-

were

series, basal parameters

sue.18 The electrodes

(Santa Clara, CA).

Measurements

methods

foil electrodes

from Sigma (St. Louis, MO). All other chemicals

when

The rate of luminal

unless otherwise

mounting

VIP and PGE, Upjohn

CA) except

as ltrnol* h-’ * cmp2, I,, in PA/

(R) was determined

and PD were allowed

by Dr. Jean

shortVCC600;

and mean values for consecutive

EFS

(The

the luminal 2.46 (267 + 2

Clamp

San Diego,

mV.

intervals

Stimulation

(Salk Institute,

1.95;

continuously

at each time point

is expressed

PD

5-minute

under

PD was measured.

secretion

Materials

Robert

4.6, and

(Voltage-Current

in each experimental

kindly provided

1.3. In

SO,‘-,

15.1, and mannitol,

Instruments,

for a lo-35-minute

were

to

CO,.

(all millimo-

130; and Mgaf, contained

were performed

on the experimental

Materials and Methods

Riviir

previ-

95% O./S%

contained

1; D-glucose,

S0,2-,

(<2 seconds)

cm2,

cholinergic

was calcu-

as validated

with

solution

conditions

bonate

noncholinergic

pathway).

relationship

411

(10 mL) was buffered

solutions

nutrient

solution

on intestinal

(i.e., an atropine-sensitive

solution and gassed

and nutrient

the

effect

that at least two different

bathing HCO,-

Both luminal

circuited

and, second,

denal mucosal

SECRETION

secretion)

of HCl infused

lars): Na+, 122; K+, 5; Ca ‘+, 2; Cl-,

first, that the

are operative

role of the enteric

(i.e., bicarbonate

the amount

pH 7.40 with

imply,

transport

The purpose

BICARBONATE

mOsm/kg).

in several

pathways

an

DUODENAL

alkalinization

The nutrient

stimulation

induced

Cl- secretion.

by atropine,

neurotoxin

lated from

Na+ and Cl- fluxes that were sug-

of active

inhibited

electrical

rabbit

and unidirectional gestive

minal

ace-

AND

ously.”

that

current

SYSTEM

The

VIP.”

showed

of stripped

(EFS)

receptors.15

cells are most probably

NERVOUS

occurred

EFS was applied rest period.

15-minute

at a fre-

of 1010 microseconds

period

for 15 min-

EFS was then followed

by an-

other 30-minute rest period. The maximal change in I, induced by EFS was measured during the second minute, whereas the peak HCO,- response occurred at 10 minutes.

412

HOGAN ET AL.

GASTROENTEROLOGY Vol. 105, No. 2

Table 1. Mean f SEM Net Peak Increases

in HCO,Secretion and I,, in Response to EFS at Varying Frequencies and Wave Durations

0.1 mV,

1.6 f

immediate

I,,, and PD (Figure

Duration

EFS period,

510 ps

respectively

and sustained

1010 ps

Frequency (Hz)

AHCO,-

AL

AHCO,-

AL

5 10 20

0.24 f 0.10 0.33 k 0.04 0.21 k 0.10

0.7 + 0.10 1.1 f 0.10 1.6 f 0.30

0.17 f 0.07 0.53 f 0.05 0.17 + 0.12

3.2 f 0.60 8.9 f 1.70 2.4 + 0.70

neurotoxin

TTX

tion stimulated (db-CAMP).

on duodenal

After

measuring (lo-’

bathing

Ten

solution.

was stimulated fect of increasing

bicarbonate

to the nutrient

PGE,,

significantly

elevated

basal for the remainder

above

fl/cm2,

when

at increasing

secretion

in separate

L), PGE,

parameters.

(10e4 mol/L),

determined. creased

VIP,

rabbit

duodenal

dose-dependent

manner.

were randomized

l.O[

0

9~‘oConditions

added The

ergic mediation stimulated CAMP).

of duodenal

401

measuring

and PD, atropine

(lop6

nutrient bathing bonate secretion

mol/L)

in-

secretion

in a

muscarinic bicarbonate

VIP, PGE,, bicarbonate

or lop5 mol/L)

cholin-

and CAMP (dbsecretion,

Isc,

was added

to the

solution. Fifteen minutes thereafter, was stimulated as described above.

bicar-

Effects of EFS and Carbachol bicarbonate secretion, Isc, and PD were - cm’, 22.4 + 2.0 PA/cm’, and

pmol/h

+

b

3.0[

20 c

40

100

120

6

60

80

EFS

100

120

EFS -

secretion

Results Resting

80

EFS 0 EFS alone 0 llX + EFS

Id

Results are expressed as means + SEM. Net HCO,and net I,, refer to peak responses minus basal. Student’s t test for paired and unpaired values was used in the statistical analyses, and P value < 0.05 was considered significant.

+ 0.10

60

tested in each series

potential

basal

40

were

significantly

Statistics

1.32

+

0

by EFS, carbachol, After

+ EFS

the effect

tissues.

mucosal

20

EFS

Effect of Atropine These studies evaluated

0 llX

..’

in bicarbonate

(lo-’

bicarbonate

and to separate

0 EFS alone

by VIP (10d6 mob’

db-CAMP

mucosal

T

of carbachol

Furthermore,

induced

and

EFS 4 c

so-

bicar-

the ef-

( lop6 to 10e4 tissues,

changes

and db-CAMP

PGE,,

EFS 4 6

2.01

bathing

stimulated

secretion

were randomized.

maximal

of TTX on the peak responses

to the nutrient

concentrations,

secretion,

I,,, and PD. Each concen-

side, and experiments

and electrical

added

secre-

to determine

of carbachol

independently

that induced

to 75.2 f 9.0 and 79.6 + 7.0

above.

effect of TTX was tested on the concentration ( low5 mol/L)

of the experi-

effect in tissue R, from a

respectively.

Carbachol, lution

(P < 0.01) and remained

EFS had no significant

basal of 74.6 XL8.1 R/cm2

the second I,,, and I’D)

to the nutrient

bicarbonate

were conducted secretion,

during

(i.e., HCO,-,

an

secretion,

and CAMP

bicarbonate

later

concentrations

was studied

the effect of the

was added

minutes

on bicarbonate

tration

basal

mol/L)

by EFS as described

experiments

mol/L)

mucosal

by EFS, VIP, carbachol,

I,, and PD, TTX

Initial

assessed

1). Furthermore,

all parameters

Effect of TTX This series of experiments

in HCO,-

again increased ment.

NOTE. Voltage, 100 V; AI,,, PA/cm*; AHCO,, umol/h . cm’; N z 4.

(n = 5). EFS induced response

0 EFS alone EFS

2.5 9 E ; 2.0 . a 1.5

-

1.01.‘::::::“.:.‘.::“:::.’ 0 20 40 Time

60

80

100

120

(Min)

Figure 1. Effects of EFS on duodenal mucosal bicarbonate secretion, lsC,and PD. EFS was applied for two 15-minute periods separated by a 30-minute rest period. EFS stimulated significantly (P i 0.01) all parameters. TTX (lo-’ mol/L), added to the nutrient bathing solution at 10 minutes, abolished EFS-stimulated bicarbonate secretion, IsC,and PD (n = 4).

ENTERIC NERVOUS SYSTEM AND DUODENAL BICARBONATE SECRETION

August 1993

bonate

secretion

in a dose-dependent

manner.

The

I,,, and PD values occurred with peak bicarbonate, lop5 mol/L carbachol; net increases above basal were - cm2 and 14.1 f 0.9 l.tA/cm’,

0.43 + 0.1 pmol/h spectively,

and PD increased

carbachol-induced sponses

to 2.3 + 0.1 mV.

increases

to EFS (Figure

ate response

were

similar

1). Because

occurred

added to the nutrient

EFS-stimulated and totally

(Figure

1). Also,

abolished

with

solution

in-

secretion

by

EFS alone

(i.e.,

R remained

I,,, and PD (Figure

decreased

bicarbonate

significantly

response

However,

TTX

induced

2). VIP also increased

the VIP-stimulated

by 73% f 17% (Figures

had no significant

increases

I

0 VIP alone 0 TTX + VIP

VIP lo-%4 $

unchanged

(P < 0.05). Of importance,

R by 13.9 -t 1.7 a/cm* TTX

(u^ c

15

the I,, and I’D responses

tissue

from basal measurements (AR = -0.25 + 1.5 C&‘cm’). VIP (1 OW6mol/L) significantly increased bicarbonate secretion,

’mito.2

re-

Secretion

bicarbonate

the

EO.4

$9

this concentra-

bathing

71% f 8% (P < 0.05) compared control)

27 0

alone

experiments.

Effect of TTX on Bicarbonate hibited

The

to the

0 VIP

the peak bicarbon-

at 10e5 mol/L,

tion was used in subsequent

TTX

re-

VIP ;o-6M

0.6

c

.-0

413

=

I

-5

peak 2 and 3).

effect on the VIP-

in I,, and PD (Figure

2

20

0

3.0

F

40

60

80

100

0 VIP alone

VIP 10% $

0 TTX + VIP

20

80

2); also, R was

unaffected. Administration

of TTX

on carbachol-, carbonate

secretion

Isc, PD, more,

PGE,-,

and

mol/L)

effect bi-

(Figure

R induced

additional

no effect

had no appreciable

and db-CAMP-stimulated 3) or on the increases by these

experiments

on a submaximal

indomethacin lutions;

that

concentration

of PGE,-stimulated

the presence

agonists.

showed

[29 l.tmol/L],

FurtherTTX

had

(3 X lo-”

bicarbonate

of full cycle-oxygenase

in

secretion inhibition

in so-

Effect of Atropine Two concentrations pine

( low5 mol/L)

secretion

completely

stimulated by about

(Figure

50%. Of note,

100

Figure 2. The effect of TTX (lo-’ mol/L) on VIP-stimulated

net bicarbonate secretion, lSc, and PD. Whereas TTX (added at 10 minutes) inhibited (P < 0.01) the VIP-induced bicarbonate response, TTX had no effect on lScand PD (n = 4).

were tested on secretion.

abolished

by EFS, whereas

by 57% + 6% (P < 0.05) creased

of atropine bicarbonate

40 60 Time (Min)

(i.e.,

added to both bathing

data not shown).

EFS- and VIP-stimulated

0.51 0

Atro-

bicarbonate

I,, was reduced

4); PD was also deat a lower

concentra-

tion of atropine (lop6 mol/L), bicarbonate secretion was decreased by 54% & 4% (Figure 3), whereas I,, was not significantly altered (<25% decrease; P > 0.05; n = 4). These findings suggest that EFS acts via a muscarinic cholinergic receptor while also releasing a noncholinergic mediator that stimulates another transport pathway. Atropine ( 10m6 mol/L) significantly inhibited VIPstimulated bicarbonate secretion and I,, by 69% k 13%

and 43% f 16%, respectively unaffected. 1O-5 mol/L

Increasing

(Figures

the concentration

did not further

decrease

3 and 5); PD was of atropine

to

the bicarbonate

and I,, responses induced by VIP; however, PD was decreased by 50%. Therefore, VIP’s action appears to involve two separate pathways: via muscarinic cholinergic receptors and directly on, or in close proximity to, the duodenal enterocytes. As expected, atropine (1 O-” mol/L) completely abolished the increases in bicarbonate secretion, I,,, and PD induced by carbachol(1 Op5 mol/L) (Figures 3 and 6). As shown in Figure 3, atropine had no significant effect on bicarbonate secretion stimulated by either PGE, or db-CAMP; the increases in I,,, PD, and R

414

HOGAN ET AL.

1.0

GASTROENTEROLOGY Vol. 105, No. 2

-Agent alone 0 Atropine DTTX

Figure 3. The effects of TTX (1 O-’ mol/L)

and atropine ( 10e6 mol/L) on net peak bicarbonate secretion stimulated by either EFS, carbachol ( 10d5 mol/L), VIP (lo-’ mol/L), PGE, (1 0m4 mol/L), and db-CAMP (lo-’ mol/ L). TTX significantly inhibited EFS- and VIPstimulated bicarbonate secretion (P < 0.01). Atropine significantly inhibited EFS-, carbachol-, and VIP-stimulated bicarbonate secretion (P < 0.01). For each series, n 2 4.

0.0 EFS

were

also unaffected

(lop5

mol/L)

by atropine. (3 X lo-9

lated bicarbonate

Comparing

analyzing

bonate

secretion

atropine, TTX

there

suggests priate changes

with I,, when

parameters

there

by bicarbonate

2)

were usually

bicarbonate was

2). This suggested

TTX

stimulated

by either

of enteric

nerves,

had no effect on either

secretion

a corresponding that the changes

transport.

However,

the neurotoxin bicarbonate increase

of

in I,

est, albeit

two transport is needed changes

differences.

Whereas

VIP-stimulated decrease

bicarbon-

significantly.

This

nerves.

tors as atropine

appro-

EFS-induced

in bicarbonate

to equate secretion.

Discussion The results from the present study show that the enteric nervous system plays an important role in the regulation of bicarbonate secretion by mammalian proximal duodenum. Furthermore, a muscarinic cholinergic receptor is activated by acetylcholine at some point, because atropine completely blocked the effect of EFS on bicarbonate secretion. Moreover, VIP stimulates bicarbonate secretion at least in part via the activation of enteric nerves, resulting in acetylcholine release. VIP-stimulated bicarbonate secretion was significantly inhibited by both TTX and atropine. In

The

was mediated

to VIP

attempting

mately stance ing

which

increase

(e.g., a neuropeptide) stimulation.

receptors

induced

that

neural

of bicarbonate

secre-

of TTX.

is unknown

The

but may functional

in bicarbonate

secretion

of the submucosal

by muscarinic

abolished

of

there was a mod-

of some residual

stimulation

55% by atropine,

muscarinic

indicated

increase

Isc response

electrical

sig-

of EFS on

the EFS

However,

of the presence

in Jsc was secondary when

PD,

blocked

to EFS in the presence

caused by electrical

periods

that was pre-

the effect

for this observation

pathways.

Therefore,

and

and

insignificant,

explanation

rones

inhibited

was involved.

tion in response

secretion

in I,, and PD. Administration

TTX secretion

stimulation

15-minute

bicarbonate

ceded by an increase

enteric

notable

inhibited

over two separate

elevated

were

in I, with

transport

was independent

and atropine

EFS given nificantly

be a result

Jsc did not

caution

because

Secretion to J,,

secretion

in both

that the increase

stimulating

bicarbonate or CAMP

the VIP-induced responses in bicarand Js,, and the effects of TTX and

and atropine

ate secretion,

PGE,

these responses.

In most cases when

in JJc were caused

db-CAMP

contrast,

of PGE,-stimu-

to net ion flux ( Jsc) (Table

or decreased, in Jsc (Table

when

mol/L)

bicarbonate

that changes

change

atropine

effect on a submaxi-

of Bicarbonate

the I,, was converted

increased

PGE2

secretion.

Comparison

comparable.

VIP

Of note,

had no significant

mal concentration

showed

Carbochol

cholinergic

the response.

Of interest,

was diminished indicating

neurecepthe

by approxi-

that another

sub-

may also be released

dur-

Further,

by carbachol

stimulation

also resulted

of in sig-

nificant increases in bicarbonate secretion, I,,, and PD, which were sensitive to atropine but not TTX. Thus, these results provide evidence that acetylcholine is released during enteric nerve activation and that it acts at muscarinic cholinergic receptors on the duodenal enterocytes to stimulate secretion. Because atropine abolished carbachol-stimulated I,, yet only partially diminished the EFS-induced I,, response, another ion(s) transport pathway, most likely Cl-, is stimulated during electrical stimulation. It has been suggested that VIP and/or PHI may function as the noncholinergic

ENTERIC NERVOUS SYSTEM AND DUODENAL BICARBONATE SECRETION

August 1993

mediator(s) tinal

ion

tion.”

responsible transport

elicited

Also, Javed

EFS released increase

for evoking

by enteric

and Cooke”

acetylcholine

reported

which

Cl- secretion),

0 VIP alone

in intes-

nerve

from guinea

in acetylcholine,

(i.e., increased

changes

415

VIP_

0 Atrooine

+ VIP

activa-

recently

that

pig colon.

The

was correlated

with I,,

was attenuated

by TTX.

In a comparative

study in bullfrog duodenum in reported that EFS significantly vitro, Crampton et a1.23 increased bicarbonate secretion, an effect that was blocked

by TTX.

in contrast increase

However,

PD was unaffected

with our results

in which

in all parameters.

disparity

is uncertain

(amphibian

The

but

0 VIP alone

1

EFS alone 0 Atropine + EFS

i .J,!,.-k

1 ,‘J.!..

I

15’ 0

20

40

80

60

100

9 E -

0 Atropine

.a.I

0.

1

1

1.0

.

./o-o I 1

0.5 t 0

20

1.0

.’ /

&-0

+ EFS

0 Atropine

+ VIP

T

b,o-tr-g,

-

E EFS alone

0

100

c 10+y6M

1.5

80 0 VIP olone

VIP 1 o-6M

2.0 : Atropine

120

60

40

1

20

+ VIP

10%

F

t 0

0 Atropine

VIP

45 r

by species

2.5

0.6

100

80

for this

be caused

‘-. ,,..!...! 0

60

40

an

EFS

.Atropine 1;-51*I

0

differences.

EFS

&A..

explanation

may

vs. mammalian)

EFS induced

’ 20

1.31

by EFS

I

?? -i-

-i-Y

/

40

..O.O.@.@

Time

60

80

100

(Min)

‘0.0 ‘\

15’ 0

-..

I

20

40

80

60

Figure 5. Effects of atropine (10m6 mol/L) on VIP-stimulated bicarbonate secretion, Ix, and PD. Atropine (added at 10 minutes) inhibited significantly both bicarbonate secretion (P < 0.01) and 1%(P < 0.0 1) and had no effect on PD (n = 4): of note, atropine (10m5mol/L) reduced PD by 50% and had no additional effect on bicarbonate secretion and lsc.

OQOQO -

100

120

??EFS

alone

3.0

i.1

2.5

o Atropine

+

EFS

VIP receptors

9

that invoke A

2.0

ent

2

study,

are present

changes the

interaction

nerves, and acetylcholine both TTX and atropine

1.5

0

20

40

60 Time

80

100

120

(Min)

Figure 4. Effects of atropine (1 Oe5 mol/L) on EFS-stimulated bicarbonate secretion, Isc,and PD. Atropine, added to the nutrient bathing solution at 10 minutes, abolished bicarbonate secretion and reduced 1%and PD by 57% and 50%, respectively (P < 0.05; n = 4).

on intestinal

enterocytes

in Cl- secretion.20’2’*24 In the presbetween

VIP,

is of interest. significantly

the enteric

Surprisingly, inhibited the

VIP-stimulated bicarbonate response by about 70%. However, neither I,, (and Jsc) nor PD were abolished completely by either TTX or atropine, suggesting alternate VIP-stimulated pathways. Thus, VIP not only stimulates ion transport directly at the enterocyte but also indirectly via the enteric nerves. VIP and acetylcholine act synergistically to mediate

416

HOGAN

ET AL.

’

GASTROENTEROLOGY

Cl- secretion in the ileum.20,25 It has been proposed that muscarinic cholinergic agents increase intracellular calcium and that VIP increases intracellular cAMP.~~ However, we have shown previously in rabbit duodenum that VIP, at doses that significantly stimulate bicarbonate secretion, may function independent of intracellular cAMP.‘~‘~ Because most of VIP’s effect on bicarbonate secretion (i.e., approximately 70%) is mediated by the enteric nerves, the present results provide additional evidence corroborating earlier observations, suggesting that separate pathways are involved in VIP- and CAMP-stimulated duodenal bicarbonate secretion. 9~‘oHowever, because VIP-stim-

0 Corbochol Corbachol 10_5M c !’

?? \:

a Atropine

alone + Corbochol

\

Yol. 105.

No. 2

Table 2. Comparison of HCO,- Transport to kc Represented as

J,, AHCO,- cm2)

(pmo//h EFS +TTX

0.49 0.13

+ 0.05 + o.03a

+ATR VIP

0.01 0.43 0.13 0.15 0.43 0.41 0.05 0.61 0.64 0.47 0.56 0.64 0.48

f + f f * & f + k f + f *

+TTX +ATR Carbachol +TTX +ATR PGE, +TTx +ATR CAMP +TTX +ATR

0.06a 0.05 0.08” 0.06’ 0.06 0.05 0.04a 0.03 0.06 0.02 0.01 0.04 0.03

@Ec$kcm2) 0.42 -0.01 0.20 0.35 0.35 0.25 0.54 0.65 0.09 0.69 0.70 0.65 0.66 0.64 0.64

f 0.10 f 0.01” f 0.02a z!z 0.03 * 0.13 + 0.04 + 0.03 -c 0.09 f o.048 + 0.10 + 0.05 + 0.06 f 0.1 1 & 0.12 f 0.05

NOTE. Bicarbonate secretion and J, measured under resting conditions were 1.80 f 0.10 umol/h . cm2 and 1.12 + 0.10 FEq/h . cm2, respectively. Values are net peak changes in ion transport. Isc was converted to J,, using Faraday’s law, i.e., multiplying the lsc by 0.0373. Concentrations of agents were as follows: VIP, 10e6 mol/L; carbachol, 10e5 mol/L; PGE,, 10e4 mol/L; db-CAMP (CAMP), 10e2 mol/L; TTX, 10m7 mol/L; and atropine (ATR), 10e6 mol/L and 10e5 mol/L with EFS. n 2 4. aP < 0.01 vs. agent alone.

20

40

60

80

Corbachol 0 Carbachol 0 Atropine

10;5u

Atropine

alone + Carbachol

i

g\

% z

0 -5: 0

20

40

Carbochol

60

80

100

ulated bicarbonate, I,, and PD responses were neither completely inhibited by TTX nor by atropine, the overall mechanism(s) of VIP-induced duodenal ion transport require additional study. Application of neural transmitters as well as neural stimulation in experimental animals significantly influences bicarbonate secretion.‘*26,27 Furthermore, in humans atropine decreased resting bicarbonate secretion by about 80%, suggesting the importance of cholinergic tone. 28 Therefore, neural mechanisms appear to have a substantial role in the regulation of mammalian duodenal mucosal bicarbonate secretion, and VIP probably acts, at least in part, via the enteric nervous system.

0 Corbochol alone o Atropine + Carbochol

1OT5M

References 1. Flemstrom

G, Heylings JR, Garner A. Gastric and duodenal HCO,transport in vitro: effects of hormones and local transmitters. Am J Physiol 1982;242:G 100-G 110. 2. Simson JN, Merhav A, Silen W. Alkaline secretion by amphibian duodenum. I. General characteristics. Am J Physiol 1981;240:

G40 1-G408. Simson JNL, Merhav A, Silen W. Alkaline secretion by amphibian duodenum. Ill. Effects of BDcAMP, theophylline, and prostaglandins. Am J Physiol 198 1;24 1 :G528-G536. 4. Flemstrom G, Garner A, Nylander 0, Hurst BC, Heylings JR. SUrface epithelial HC03(-) transport by mammalian duodenum in vivo. Am J Physiol 1982;243:G348-G358. 5. lsenberg JI, Flemstrom G. Johansson C. Mucosal bicarbonate secretion is significantly greater in the proximal versus distal duo3. 20

40

Time

60

80

100

(Min)

Figure 6. The effect of atropine ( 10e6 mol/L) on carbachol-stimulated bicarbonate secretion, lsc, and PD. Atropine (added at 10 minutes) abolished all parameters stimulated by carbachol (P < 0.0 1; n = 4).

August 1993

ENTERIC NERVOUS SYSTEM AND DUODENAL BICARBONATE SECRETION

denum in the in vivo rat. In: Allen A, Flemstrom G, Garner A, Silen W, Tumberg LA, eds. Mechanisms of mucosal protection in the upper gastrointestinal tract. New York: Raven, 1984: 175- 180. 6. Vattay P, Feil W, Klimesch S, Wenzl E, Starlinger M, Schiessel R. Acid stimulated alkaline secretion in the rabbit duodenum is passive and correlates with mucosal damage. Gut 1988;29:284290. 7. Konturek SJ, Bilski J, Tasler J, Laskiewicz J. Gut hormones in stimulation of gastroduodenal alkaline secretion in conscious dogs. Am J Physiol 1985;248:G687-G691. 8. lsenberg JI, Hogan DL, Koss MA, Selling JA. Human duodenal mucosal bicarbonate secretion. Evidence for basal secretion and stimulation by hydrochloric acid and a synthetic prostaglandin E, analogue. Gastroenterology 1986;9 1:370-378. 9. Yao B, Hogan DL, Bukhave K, Koss MA, lsenberg JI. Bicarbonate transport by rabbit duodenum in vitro: effect of vasoactive intestinal polypeptide, prostaglandin E,, and cyclic adenosine monophosphate. Gastroenterology 1993; 104:732-740. 10. Yao BG, Hogan DL, Koss MA, lsenberg JI. Transport pathways involved in rabbit duodenal mucosal bicarbonate secretion (DMBS) in vitro (abstr). Gastroenterology 199 1; 1OO:A7 10.

with electrical field stimulation 20.

21.

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24.

25.

11. Furness JB, Bornstein JC. The enteric nervous system and its extrinsic connections. In: Yamada T, ed. Textbook of gastroenterology. Volume 1. Philadelphia: Lippincott. 199 1:2-24.

26.

12. lsaacs PET, Corbett CL, Riley AK, Hawker PC, Turnberg LA. In vitro behavior of human intestinal mucosa. The influence of acetylcholine. J Clin Invest 1976;58:535-542. 13. Coupar IM. Tetrodotoxin inhibits directly acting stimulants of intestinal fluid secretion. J Pharm Pharmacol 1986;38:553-555.

14. Llewellyn-Smith IJ, Furness JB, O’Brien PE, Costa M. Noradrenergic nerves in human small intestine-distribution ture. Gastroenterology 1984;87:5 13-529.

and ultrastruc-

15. Jodal M. Neuronal influence on intestinal transport. J Intern Med 1990;228(Suppl 1): 125- 132. 16. Hubel KA. The effects of electrical field stimulation and tetrodotoxin on ion transport by the isolated rabbit ileum. J Clin Invest

1978;62:1039-1047. 17. Perdue MH, Davison JS. Altered regulation of intestinal ion transport by enteric nerves in diabetic rats. Am J Physiol 1988;254:G444-G449. 18. Cooke HJ. Influence of enteric cholinergic neurons on mucosal transport in guinea pig ileum. Am J Physiol 1984;246:G263267. 19. Hubel KA, Shirazi S. Human ileal ion transport in vitro: changes

27.

28.

and tetrodotoxin.

417

Gastroenterol-

ogy 1982;83:63-68. Cooke HJ, Zafirova M, Carey HV, Walsh JH, GrinderJ. Vasoactive intestinal polypeptide actions on the guinea pig intestinal mucosa during neural stimulation. Gastroenterology 1987;92:36 l370. Hubel KA, Renquist KS, Varley G. Noradrenergic influence on epithelial responses of rabbit ileum to secretagogues. Am J Physiol 1989;256:G9 19-G924. Javed NH, Cooke HJ. Acetylcholine release from colonic submucous neurons associated with chloride secretion in the guinea pig. Am J Physiol 1992;262:G 13 1-G 136. Crampton JR, Gibbons LG, Rees WD. Neural regulation of duodenal alkali secretion: effects of electrical field stimulation. Am J Physiol 1988;254:G 162-G 167. Dharmsathaphorn K, Harms V, Yamashiro DJ, Hughes RJ, Binder HJ, Wright EM. Preferential binding of vasoactive intestinal polypeptide to basolateral membrane of rat and rabbit enterocytes. J Clin Invest 1983;7 1:27-35. Dharmsathaphorn K, Pandol S. Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line. J Clin Invest 1986;77:348-354. Lenz HJ, Vale WW, Rivier JE. TRH-induced vagal stimulation of duodenal HCO;-mediated by VIP and muscarinic pathways. Am J Physiol 1989;257:G677-G682. Nylander 0, Flemstrom G, Delbro D, Fandriks. Vagal influence on gastroduodenal HCO; secretion in the cat in vivo. Am J Physiol 1987;252:G522-G528. Ballesteros MA, Wolosin HD, Hogan DL, Koss MA, lsenberg JI. Cholinergic regulation of human duodenal mucosal bicarbonate secretion. Am J Physiol 199 1;26 1:G327-G33 1.

Received November 9, 1992. Accepted March 16, 1993. Address requests for reprints to: Jon I. Isenberg, M.D., Division of Gastroenterology (8413), University of California at San Diego Medical Center, 225 Dickinson Street, San Diego, California 921038413. Supported in part by National Institutes of Health Grant AM33491. The authors thank Drs. Kim Barrett and Klaus Bukhave for expert advice. Dr. Yao was a visiting scholar from the Peoples Republic of China. Portions of these studies were published In abstract form in (Gastroenterology 1992;102:A255).