Vol.

170,

No.

July

16, 1990

1, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

Pages

MODE OF STIMULATORY ACTION OF DEOXYCHOLATR IN SIGNAL SYSTEiM OF ISOLATED RAT PANCREATIC ACINI'

HIROYUKI

NAKANISHI, YOICHI

t4ay 14,

111-118

TRANSDUCTION

YOSHIFUMI TAKEYAMA', HARUMASA OHYANAGI, SAITOH, AND YOSHIMI TAKAI*

Departments of Surgery (1st Division) Kobe University School of Medicine, Received

COMMUNICATIONS

and *Biochemistry, Kobe 650, Japan

1990

Mode of stimulatory action of deoxycholate (DCA) on the secretaglogue-induced amylase release and the phospholipase C reaction in isolated rat pancreatic acini was investigated using activator of GTP-binding sodium fluoride (NaF), which is a direct DCA enhanced the amylase release induced proteins: (G proteins). by submaximal concentrations of NaF without affecting the maximal level of this reaction. Under the similar conditions, DCA enThese stimuhanced the NaF-induced phospholipase C reaction. latory effects of DCA on the NaF-induced amylase release and phospholipase C reaction are comparable to those on the secretagogue-induced reactions reported previously. These results suggest that DCA acts on the coupling of a G protein(s) to the phospholipase C in the membrane transduction mechanism in isolated rat pancreatic acini. 0 1990Academic mess, Inc.

1The investigation in the Department of Surgery (1st Division) was supported by Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (1989). The investigation in the Department of Biochemistry was supported by Grants-in-Aid for Scientific Research and Cancer Research from the Ministry of Education, Science and Culture, Japan (1989), Grants-in Aid for Abnormalities in Hormone Receptor Mechanisms, Cardiovascular Disease and for Cancer Research from the Ministry of Health and Welfare, Japan (1989), and by Grants from the Yamanouchi Foundation for Research on Metabolic Disease (1989) and the Research Program on Cell Calcium Signal in the Cardiovascular system (1989). 2To whom requests for reprints should be addressed. Abbreviations used are: DCA, deoxycholate; CCKB, cholecystokinin-octapeptide; G protein, GTP-binding protein; NaF, CCK, cholecystokinin; Hepes, N-3-hydroxylsodium fluoride; '-2-ethanesulfonic acid. ethyl-piperazine-N

111

0006-291X/90 $1.50 Copyright 0 1990 by Academic Press. Inc. Ail rights of reproduction in any form reserved.

Vol. 170, No. 1, 1990

A preceding new action their

of bile

from

laboratories

acids

action

of

presumably

involved

titis.

is

Low concentrations

sensitize

the

pancreatic

DCA potentiates

release.

amylase

phosphoinositide

hydrolysis

and thereby ing

of

enhances

CCK8 to

subsequent

its

Ca2+ mobilization

enhances

amylase

the

but

phospholipase the

suggests

receptors

adenylate

to the cyclase

NaF can mimic

In amylase

this release

pancreatic DCA in

acini. the

Mode of

action

for

the

secretagogue-induced

phosphoof DCA on

phospholipase

involved

the

coupling

with

has recently (15,16).

action

of the

in

by analogy

in

C reaction

of the

been has amylase acini

(7).

NaF-induced isolated

stimulatory C

the

It

CCK in

pancreatic in

phospholipase

C

Accumulating

DCA enhances

action

DCA

to be clarified.

the

and phospholipase

that

of the

stimulatory

C reaction

we show that

intra-

indicated

enhancement

of G proteins

phospholipase

paper,

has been

bind-

processes

and the

Fluoride

(13,14).

been

and the

the

C (4-12)

activator

release

affecting

is

phospholipase

shown that

C-catalyzed

investigated.

shown to be a direct also

CCK8-induced

without

a G protein(s)

system

the

acid,

secretagogue

mechanism

intensively

that

bile

pancrea-

by the

remains

regulatory

has been

acute

induced

the

mode of

C reaction

Recently, evidence

through the

in

or secretory

It

is

pancreatitis

C activation

(1).

release

C reaction,

by receptors

kinase

This

phospholipase

on acini

protein

cellular lipase

release

receptors

to the

is

from

pancreas

entity

the

which

amylase

(2,3).

exocrine

and enhance

a

different

DCA , a secondary

acini

has reported

of biliary

a major

of

(1) pancreas

on the

development

which

RESEARCH COMMUNICATIONS

previously

acids

the

cholelithiasis

exocrine

described

bile

in

our

on the

effects

stimulatory

of

AND BIOPHYSICAL

report

destructive

due to

BIOCHEMICAL

rat effect

reaction

of is

dis-

cussed. MATERIALS

AND METHODS

Materials and chemicals employed were purchased as described previously (1). Isolated pancreatic acini were prepared from a starved male Wistar rat (200-250 g) by the method of Williams (17). Amylase release was assayed as described previously (1). Amylase activity was determined also as described previously (1). The amylase release was expressed as the ratio of the value of the amylase activity released into the medium during the incuba112

Vol.

170,

No.

1, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

tion to that of the total amylase content. The total amylase content was estimated by measuring the enzymatic activity of a l-ml aliquot of the unstimulated acinar suspension after disruption of the acinar cells by sonication. Diacylglycerol and inositol phosphate formati ns were assayed as described previousRadioactivity of 9 H-labeled sample was determined using ly (1). a Beckman liquid scintillation system, Model LS 3801. RESULTS Effect

of

Figure

1A shows the

release.

DCA on the

Incubation

release

in

effects

were

of

NaFeffect the

of

acini

a dose-dependent observed

and CCK8-induced DCA on the with

Amylase

Release

NaF-induced

NaF induced

amylase

marked

amylase

manner.

The maximal and half maximal 10 mM and 5 mM of NaF, respectively.

with

II

A

I-

10

5

NaF

(mM1

15

I 0

I -11

CCKs

I -10

(log

I -9 M)

02

O

I

I

I

5

10

15

NaF

(mM)

Fig. 1. Effect of DCA on the NaF- or CCKB-induced amylase relea,se. The acini were incubated for 20 min at 37OC with various doses of NaF or CCK8 in the presence or absence of 0.25 mM DCA. (A), with various doses of NaF. ( l ), in the presence of DC.A. (0 1, in the absence of DCA. (B), with various doses of CCK8. (0 ), in the presence of DCA. ( o ), in the absence of are the means + SE of 3 independent experiments. DCA. Results Fig. 2. Effect of DCA on the NaF-induced fogmation of diacylglycerol. The acini prelabeled with [ Hlarachidonic acid as described in "Materials and Methods" were incubated with 10 min at 37OC with various doses of NaF in the presence or absence of 0.25 mM DCA. (01, in th e presence of DCA. ( 0 1, in the absence of DCA. Results are the means + SE of 3 independent experiments.

113

Vol.

BIOCHEMICAL

170, No. 1, 1990

These

results

Matozaki

are

et al.

during

the

shifted

to

(7).

incubation left. doses

reaction

The

in

the

curve

of

the

manner effects

DCA affected

was shifted

to

These

the

results

mode of

by NaF is induced

basically of

of

diacylglycerol acini

Figs.

the

acini

with

the

formation

incubation

of diacylglycerol

However,

only

slightly. (1).

release

amylase

by submaximal reactions

doses induced

the

phosphates

reactions

The

induced

release

were

our

of

laboratories in

the

formation

i.e., DCA enhanced the inositol bisphosphate, formation

of

inositol

of manners of

DCA to

enhancement

phosphates

DCA showed

a little

of NaF.

of diacylglycerol

nor

effect

DCA alone that of

used in these experiments. formation of diacylglycerol to (l),

those except

trisphosphate. we described of

the

doses

comparable

(11,

formation

dose-dependent

NaF caused

by maximal

previously

inositol

C Reaction

The addition

of NaF.

formation

as reported

formation

in

the

and inositol

phosphates in the doses of DCA on the NaF-induced

the

dose-

results

NaF caused

with

inositol effects

actions

CCKB.

Phospholipase

phosphates

neither

from

the

enhanced

amylase

on the

NaF-induced

induced

the

of

previous

2 and 3, respectively.

during

on these

inositol

our

same as that

and inositol

as shown in

induced

CCK8,

release

of DCA on the

DCA on the

Incubation

of the

mM DCA was

by CCKB.

Effect

the

the

The -10 M 1 x 10

and DCA markedly amylase

effect

@.

with

with doses

induced

amylase

Fig.

observed

incubation

with

effect

was

a little

When 0.25

of

consistent

stimulatory

were

level

curve

of NaF.

as shown in

left

acini

CCK8 induced

by submaximal

maximal

are

doses

respectively. the

induced

DCA showed

with

the

release

amylase

maximal

acini

during

release

amylase

However,

by

to

dose-response

enhanced

by the

and half maximal and 3 x 10 -11 M of CCK8,

response

NaF,

NaF.

induced

acini

reported

with

a dose-dependent

to the

results

mM DCA was added

maximal

added

the

RESEARCH COMMUNICATIONS

When 0.25

of

incubation

release

with

DCA markedly

by submaximal on this

consistent

AND BIOPHYSICAL

three

species

In that of

on the the

The and

CCKB-induced

effect

of DCA on

the previous report DCA had differential inositol

phosphates,

formations of inositol monophosphate but did not show a significant effect trisphosphate 114

induced

by the

and on

secreta-

Vol.

170,

No.

BIOCHEMICAL

1, 1990

I

I

AND

BIOPHYSICAL

RESEARCH

I

I

COMMUNICATIONS

I

I

Fig. 3. Effect of DCA on the NaF-induced forrgation of inositol phosphates. The acini prelabeled with myo-[ Hlinositol as descr.ibed in "Materials and Methods" were incubated for 10 min at 37OC with various doses of NaF in the presence or absence of 0.25 mM DG4. (A), inositol monophosphate; (B), inositol bisphosphate; (c), inositol trisphosphate. (0 ), in the presence of DCA. (0), in the absence of DCA. Results are the means + SE of 3 independent experiments.

L 9

“i0

0

-10

-11 CCK8

(log

0

I -11

I -10

I -9

M)

of DCA on the CCK8-induced fqrmation of inositol Fig. 4. Effect The acini prelabeled with myo-[ Hlinositol as phosphates. described in "Materials and Methods" were incubated for 5 min at 37Oc ,with various doses of CCK8 in the presence or absence of (A), inositol monophosphate; (B), inositol 0.25 :mMDCA. trisphosphate. (0 1, in the presence bisphosphate; (Cl, inositol ( o ), in the absence of DCA. Results are the means + SE of DCA. of 3 independent experiments. 115

BIOCHEMICAL

Vol. 170, No. 1, 1990

This

gogue. mental

discrepancy

conditions;

were

incubated

report

(11,

for

in

ed acini

were

formation

of

other

acini

1.5

min

whereas

10 min

was derived

the for

the

this

prelabeled with

paper.

the

RESEARCH COMMUNICATIONS

the

different

with

acini

CCK8 for

5 min,

trisphosphate

in

were Fig.

experi-

myo-['HI-inositol

secretagogue

As shown in with

inositol

from

prelabeled

incubated

inositol

AND BIOPHYSICAL

the

previous

incubated

with

4, when the

prelabel-

DCA enhanced

as well

as the

NaF

the

formation

of

phosphates. DISCUSSION

Our previous gogue-induced amylase

release.

However,

the

In

(12,131. form

the

effect

comparable effect

to

of

to

on the

to the the

that

with processes

coupling

are of the

three

of

the

to the

the G protein(s) to the phospholipase C-catalyzed phosphoinositide hydrolysis. sites, amylase

the

release

by CCK8 but protein(s).

first

one is and the

also It

less

possible

phospholipase

by NaF which is

possible

(1)

coupling

sites

is that

of the

of

release

is The is

also

CCK8 receptor

phosphoinositides

action

by

system,

of DCA, i.e.,

G protein(s), C, and the

coupling

of

phospholipase

Among these three because DCA enhanced the C reaction

a direct

activator

DCA acts

on the

116

doses

These observasuggest that DCA

transduction of

DCA

C reaction

reaction.

of

acini

release.

amylase

phospholipase

membrane

CCK8 receptor

that

amylase

in

to the

submaximal

NaF-induced

results

fluoride,

pancreatic

by the

hydrolysis

possible

CCK in

CCK

cells

coupled

induced

previous

the

acinar

demonstrated

CCK8-induced

between

C in

of the

shown that

CCK8-induced

and the

phospholipase

coupling

we have

NaF-induced our

the

has been

G protein(s)

action

DCA on the

on the

G protein(s) There

the

on the

that

together

acts

of

also

the

release

DCA on the

comparable tions

has been

potentiates it

C in pancreatic

study,

amylase

in

secreta-

of DCA on the

Recently,

involved

can activate

present

the

This

NaF.

it

C and mimic

In

enhances

is

the

and thereby

mode of action

phospholipase

of NaF,

DCA enhances

was unclear.

addition,

phospholipase (12) -

the

a G protein(s) to

that

C reaction

C reaction

shown that receptor

showed

phospholipase

phospholipase

the

report

induced of coupling

not the

only

G of

the

Vol.

170,

No.

BIOCHEMICAL

1, 1990

fluoride-sensitive enhances the

G protein(s)

the

phospholipase

subsequent

protein(s) creas

the

may be different (71,

but

has not

been

on the

coupling

On the

completely

neglected

hydrolysis

of phosphoinositides.

of detergents stimulates

the the

However,

enzyme

addition,

tion

in

of

much higher

NaF-induced possible

vivo

that

tein(s)

conclusion, and the

secretageogue-induced

pan-

does

in

that

DCA acts

coupling

is

be

phospholipase effect

C-induced of many types

well

in vitro has been -~ established that DCA

in

vitro

in --in used in

(18-21).

that

our

DCA used

in vivo -~ experiment

our

(data --in vitro the phospholipase

elicit

DCA enhances

C reaction. the

affinity

and enhances

the

on the

phospholipase

in

only

Thus,

it

of

the

process

does

shown).

CCK8- or less

phospholipase

the

the

G pro-

enhances pancreatic

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BIOCHEMICAL

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RESEARCH

COMMUNICATIONS

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