Electrochemical response patterns to histamine, bombesin, and pentagastrin in isolated bullfrog gastric mucosa

BIOCHEMICAL

Vol. 103, No. 4,1981 December

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1186-1193

31, 1981

ELECTROCHEMICAL RESPONSE PATTERNS TO HISTAMINE,

BOMBESIN, AND

PENTAGASTRIN IN ISOLATED BULLFROG GASTRIC MUCOSA Amram Ayalon, Phillip

M.D.,*

L. Rayford,

Peter

P.D.,***

G. Devitt,

F.R.C.S.,**

and James C. Thompson,

Department of Surgery The University of Texas Medical Galveston, Texas 77550 Received

November

M.D.

Branch

6, 1981

SUMMARY Histamine, bombesin, and pentagastrin produced different patterns of changes in short circuit current, electric conductance, potential difference, and acid secretion in isolated bullfrog gastric mucosa. Histamine produced a gradual increase in electric conductance, parallel to the increase in acid secretion, and a transient rise in short circuit current. Bombesin induced an abrupt increase in electric conductance and in short circuit current, which peaked after 8 minutes. Pentagastrin also produced an increase in short circuit current, which peaked after 8 minutes; electric conductance, however, rose more gradually. Bombesin produced only a short term increase in acid secretion. These experiments show that histamine, bombesin, and pentagastrin affect gastric mucosa by different mechanisms. Histamine may have a more pronounced effect on the fusion process and activation of the tubulovesicular system of the parietal cell; bombesin may act by transiently increasing the permeability of the basolateral membrane. Pentagastrin seems to have an effect on both the basolateral membrane and the tubulovesicular acid secretory apparatus. These observations are not consistent with the hypothesis that histamine is the final common mediator for the effects of other secretagogues. Stimulation

of acid

secretion

mucosa has been shown to cause (G), returns

an initial

*

***

potential

a sustained

in the short

to prestimulation

transmucosal

**

increase

by histamine

levels), difference

in isolated

increase

circuit

(PD)

(with

in electric

current

and an initial

frog

(Isc)

transitory

a subsequent

gastric conductance

(which increase decrease

then in

to levels

Visiting Scientist from Hadassah University Hospital, Jerusalem, Israel. Visiting Scientist from University of Bristol, Bristol, England. Recipient of a Wellcome Research Travel Grant. Present Address: Royal Liverpool Hospital, Liverpool, England. Present Address: Department of Physiology and Biophysics, University of Arkansas Medical School, Little Rock, Arkansas,

0006-291X/81/241186-08$01.00/0 Copyrighr 0 I981 by Academic Press, Inc. All rights of reproduction in any form reserved.

1186

Vol. 103, No. 4,1981

lower

than

caused frog,

BIOCHEMICAL

the prehistamine

an increase the

gastric

steady

mucosa

In frog

cause

an increase below

state

electric

suggests this final

after

gastric

mucosa,

study,

that

observation

patterns

induced

increase

in PD.

In contrast

to the

of the PD was elevated sustained

as after

rise

increase

have also

with

(2).

pentagastrin

and pentagastrin

of changes

a subsequent

is not for

gastric

affects

in acid

mucosa.

the parietal

consistent the effects

with

secretion

bombesin,

by histamine,

bullfrog

each agent

common mediator

histamine

In in PD

stimulation been drop

shown to in PD to

(5).

the

in isolated

mucosa,

as well

tetra-

COMMUNICATIONS

gastric

a similar

maculatus,

histamine,

RESEARCH

In piglet

level

in G and a transitory

measurements

determined

(1).

posthistamine

prestimulation

In this

BIOPHYSICAL

in G and a transient

has been described

levels

ones)

from Necturus

(3).

AND

and pentagastrin

Comparison cell

by different

the hypothesis of other

MATERIALS

and in the

that

of these

were patterns

mechanisms; histamine

is

the

secretagogues.

AND METHODS

Bullfrogs (Rana catesbeiana) were doubly pithed. The stomachs were removed and the serosa and muscularis were stripped away by sharp dissection. The fundic with an exposed surface area of 1.5 mu'i osa was mounted in Ussing-type chambers, with gas-lift circulating systems at room temperature. cm , and were perfused The Ringer's solution bathing the mucosa was bubbled with 100% O2 and contained the following (in mM/L): Na-110, K-5, Mg-1, Ca-2, Cl-121, and dextrose-lo. The serosal solution contained tris buffer (10 mM/L; pH 7.4). PD was measured by two saturated calomel electrodes and current was applied by Ag-AgCl electrodes, both through 4.4 M NaCl-Agar bridges. All tissues were short circuited and I was recorded on a Grass model 7 polygraph. For measurement of the open c&Sit PD, I was disconnected for 10 seconds; G was determined by applying pulses of ~1:: and minus 10 mV for 4 seconds in each direction, and measuring the resulting changes in current. Acid secretion was determined by continuous titration of the mucosal solution to pH 7.0 with 0.003 N NaOH, by an automatic titrator (Radiometer, Copenhagen). The amount of titrant added was recorded every 6 minutes. After the tissues were mounted and wese allowed to stabilize, spontaneous acid secretion dropped to below 1.7 uEq/cm hr after about 2 hours. At this stage, histamine, bombesin nonapeptide (Farmitalia, Milan), or pentagastfhn was applied to the serosal side of the tissue at a final concentration of 10 M. Results are expressed as the mean plus or minus one standard error. The Student's 't' test was used to analyze the data for statistical significance of differences between means. Differences with a ~value of less than 0.05 were considered significant.

1187

. ,I/- :I-”

Vol. 103, No. 4,198l

BIOCHEMICAL

AND

BIOPHYSICAL

l

RESEARCH

COMMUNICATIONS

l

l

l

*

G

6.2 5.6

*

5.4

3.4

i 1 -6

Figure

1.

0

6

30

12

16

24

30

36

42

46

54

60

MlNUTES

Short circuit current (I ), conductance (G), potential differ-e ce (PD) and acid secretion gfter the application of histamine (10%) * = significantly different from

RESLJLTS histamine state

level

duration after

of about

1) caused

12 minutes

increased level

and then

and then to 3.8x0.3 for

a gradual

6 mmho/cm2 after

of the experiment.

4-8 minutes,

state

(Fig

Isc

uEqlcmL

the duration

of the experiment.

1188

level

PD increased

prestimulation 18 minutes

reached

and was sustained

to a maximal

subsided.

to below hr after

in G which

24 minutes

increased

gradually

dropped

increase

for

of 114x10

the pA/cm2

in the initial

levels. and then

a steady

Acid

remained

secretion at a steady

Vol. 103, No. 4,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

MINUTES

Figure

2.

Short (PD)

After peaked

current secretion

(I

to the serosal

side,

n=5.

prestimulatory

level.

bombesin

circuit and acid

(Fig

8 minutes.

at

Z),

G rose

level

of about

5 mmho/cm2.

initial

rise

in PD was noted

12 minutes.

Acid

sharp to

Isc

conductance (G), the application * = significantly

increases

5.720.3 rose

which

secretion

), ffter

in G and Isc were

mmho/cm', to 14427

returned

increased

potential differgnce of bombesin (10 different from

and then

VA/cm'

stabilized

and then

to prestimulation

to 2.7tO.3

nEq/cm2

noted

M)

which at a

subsided. levels

An after

hr and then

gradually

subsided. Pentagastrin minutes. G increased the duration

I SC rose

(Fig

3) caused

to 15329

more gradually of the

a sharp

uA/cm',

and then

and stabilized

experiment.

increase

PD

initially

1189

in Isc which

returned

at a level increased

peaked

to prestimulation of about

at 8 levels.

4.8 mmho/cmL for

to above,

and subse-

Vol. 103, No. 4,1981

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS

5.8

5..

5 ! t

5.0

b

l.6

f

B

-4

E L1 l.2

-4 3.0

-3 3.4

-3 3.0

Figure

(G), potential difference Short circuit current (I ), conductance (PD) and acid secretion !fter the application of pentagastrin (10%) to the serosal side, n=6. * = significantly different from prestimulatory level.

3.

quently

decreased

4.520.1

LlEq/cm2 hr. In four

which

Isc

to below

membranes

dropped

biphasic

response

duration

with

which

prestimulation

were

levels.

mounted

for

Acid

periods

secretion

of over

to levels

of 14.9t2.1

uA/cm’,

pentagastrin

(Fig

Isc

showed

an initial

transient

4).

a subsequent

second

increase

which

increased

8 hours, produced rise

to

and in a clear

of short

was more gradual.

DISCUSSION These histamine,

experiments bombesin,

show that or pentagastrin

stimulation produces

1190

of bullfrog different

gastric

mucosa with

electrochemical

res-

Vol. 103, No. 4,1981

BIOCHEMICAL

AND BIOPHYSICAL

PENTAGASTRIN

Figure

ponse

4.

increase

Histamine

in acid hand,

stabilizes

a short

an abrupt

greater

lasting

rise

The increase observed

the

increase

induced

by fusion

Bombesin, been acinar output.

shown cell It

with

area

with

(2).

to cause

of the

membranes,

term

reduction

has been suggested

on the

peak after after

8 minutes,

more gradually

and

Although

the changes

pentagastrin,

the

histamine.

the

Bombesin

increase

is

apical with

acetylcholine, short

peaks

rises

after

well

The increase

of tubulo-vesicles

like

than

histamine,

mucosa

in surface

I SC which

12 minutes. than

Bombesin

SC'

to the

in

latter

produced

only

secretion.

in G, correlated

gastric

in I

G, however,

rate

in G, parallel

in Isc which

histamine

in acid

rise

a rise

after

secretory

upon stimulation

and mammalian

state

after

acid

increase

in G and in

by bombesin.

at a new steady

a higher

increase

produces

produced

G were markedly

a gradual

and a transient

Pentagastrin to that

produced

produces

secretion,

induces

8 minutes. similar

36

Shof& circuit current after the application of pentagastrin (10 M) to the serosal side of four membranes which were mounted for periods of over 8 hours, in which I dropped to low prestimulatory levels. * = significantly diffferent from prestimulatory levels.

patterns.

other

COMMUNICATIONS

IO-“,.,

12 24 MINUTES

0

RESEARCH

in acid

known in both

this

that

the change

1191

of the oxyntic

membrane

cholecystokinin,

of membrane

amphibian

in G has been attributed

membrane

depolarization

secretion,

to

cell,

(6.7).

and pentagastrin,

have

of rat

and mouse pancreatic

resistance,

and a rise

in cell

(1)

membrane

in amylase

conductance

is

Vol. 103, No. 4,198l

BIOCHEMICAL

due to an increase Cl and outflux of acinar early

suggests

implies

that

an increase

temporary

The early the clear

levels

Biphasic

effects

caerulein, mucosa

effect

of pentagastrin

of Isc

suggest

have also

We did

pentagastrin,

probably

In isolated

parietal

Moreover,

(10).

parietal

of histamine cholinergic

calcium.

Although it

cell their

is

linked

the action

was not

found

with

linked

of gastrin

to be associated

with

membrane.

the

gastric

increase

After

in acid

secretory

apparatus.

and gastrin

have been

to act

on a specific

seems to be different. AMP formation,

influx

of extracellular

shows some dependence with

low pre-

bombesin.

of cyclic

to enhanced

and

pentagastrin,

each appearing of action

peak

secretion.

of pentagastrin

carbachol,

to stimulation is

acid

and Necturus

on the acid

directly, mechanism

initiate

carbochol,

in parallel

of K with

the observed

in G, as after

histamine,

stimulation

enhanced

on extracellular

Influx

of extracellular

in isolated

gastric

(11,lZ).

Our observations mucosal

cells.

greater

effect

system,

reflected

increase

fashion

after

The

inducing

Outflux

on the basolateral

changes

an effect

mucosa,

in membranes

in frog

early

cells,

receptor The effect

find

reflecting

the

the

observed

more gradually

shown to affect

calcium

an effect

been

not

G rose

secretion,

calcium,

on Isc

(8).

of bombesin

explain

administration

and cholecystokinin-octapeptide (5.9).

whereas

after

could

uncoupling

concentration

membrane.

K might

of Na and

electric

in gastric

calcium

observed

evoke

application

effects

in intracellular

in Isc

biphasic

stimulatory

drop

COMMUNICATIONS

an influx

calcium

of the basolateral

in intracellular

peak

agents

by us after

similar

in the conductance

An increase

in Is=.

produces

these

causing

in cytosol

in G observed

bombesin

a consequent

that

an increase

term rise

RESEARCH

Na, and K conductances

The finding

of K.

cells

short

in Cl,

AND BIOPHYSICAL

similar

are consistent

histamine,

by stimulating

on the fusion by the

in acid

with

process

sustained

secretion.

to carbachol,

the

findings

cyclic

AMP formation,

and activation high-level

Bombesin by transiently

1192

increase may affect increasing

of the

may have a

tubulovesicular

in G, correlated the parietal

cell

the basolateral

with in a membrane

Vol. 103, No. 4,198l

BIOCHEMICAL

permeability,

reflected

are different

from

in Isc,

the effects

secretion.

secretion

than

histamine,

histamine

than

after

the oxyntic suggested, sites

(13).

could

thus

with the

cell changes

parietal

system,

Since

pentagastrin increase

pentagastrin,

and acid

it

secretion

in G may involve

The effects be different.

the hypothesis

tubulovesicular

that

of histamine

reflected

in the gradual

induced

a higher

in G was markedly appear

that

obligatorily

changes

in both

functions

Pentagastrin reflected

are not

that

of pentagastrin

membrane,

these

on these

observations

as the final

area

early

rise of acid

greater

structural

surface

by the

rate

coupled.

and pentagastrin

We conclude histamine

would

The effects

COMMUNICATIONS

and of bombesin.

the basolateral

and the

RESEARCH

peak in G.

of histamine

on both

and on the

in G and in acid

BIOPHYSICAL

by the early

seems to have an effect peak

AND

after changes

in

As previously and in pump two factors

are not

consistent

common stimulant

for

cell. ACKNOWLEDGMENTS

This work was supported by grants from the National Foundation, Inc. (AM 15241) and the John A. Hartford

Institutes

of Health

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Rehm, W.S. (1962) Am. J. Physiol. 203:63-72. (1975) J. Physiol. 244:15-31. Forte, J.G., Forte, T.M., and Machen,yE. Nakajima, S., Shoemaker, R.L., Hirschowitx, B.I., and Sachs, G. (1970) Am. J. Physiol. 219:1259-1262. Shoemaker, R.L., Eschowitz, B.I., and Sachs, G. (1967) Am. J. Physiol. 212:1013-1016. Nakajima, S. In: Gastric Secretion, edited by G. Sachs. New York: Academic Press, pp 225-236. Machen, T.E., Clausen, C.. and Diamond, J.M.. (1977) Gastroenterology 73:970. zdar, A.W. (1965) Fed. Proc. 24:1360-1367. Iwatsuki, N. and Petersen, O.H. (1978) J. Clin. Invest. 61:41-46. Nakajima, S., Hirschowits, B.I., Shoemaker, R.L., and Sachc G. (1971) Am. J. Physiol. 221:1009-1013. (1978z. Clin. Invest. 61:370-380. Soll, A.H. Soll, A.H. and Wollin, A. (1979) Am.?. Physiol. 237:E444-E450. Soll, A.H. and Ferrari, J.C. (in press) J. Clin. 1%&t. Forte, T.M. and Forte, J.G. (1981) Gastroenterology Black, J.A., -81:509-519.

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