The stimulation of hepatic adenylate cyclase by prostaglandin E1

Vol. 55, No. 2,1973

BIOCHEMICAL

THE STIMULATION

OF

HEPATIC Floyd

ADENYLATE

W.

Sweat

Department University Salt

Received

September

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CYCLASE

and

Thomas

of Biological of Utah College Lake City, Utah

J.

BY PROSTAGLANDIN

E,

Wincek

Chemistry of Medicine 84132

22,1973 SUMMARY

Adenylate cyclase activity associated with particulate preparations from rat, mouse, rabbit, and dog liver is stimulated 2- to 5-fold by prostaglandin ET (PGET). This stimulation is dependent upon the presence of guanosine+‘-triphosphate (GTP). Prostaglandins FTa and F2a do not alter the enzymatic activity under these same conditions. Optimal concentrations of PGET + GTP stimulate rat liver adenylate cyclase more than glucagon alone, but less than glucagon + GTP. Activity measured with glucagon + GTP is not affected by addition of PGEt . Stimulation from PGET + GTP is increased by glucagon to the same level measured with glucagon + GTP. In liver, their

ability

(1).

The

the

to activate

precluding

glandin

Many

effect

on

In this

paper,

homogenates

by

comparison

liver

, the

the

activation

salt),

cyclic

purchased Upjohn

kinase AMP

from Co.,

England

Nuclear

Merck.

Rats

mongrel Copyright All rights

dogs

(Type

(sodium

Sigma

GTP Co.

Dowex g) were

obtained

described

0 1973 by Academic Press, Inc. of reproduction in any form reserved.

the

activation measured

by

cyclic

AMP

tissue

prepara-

Furthermore,

a prosta-

previously. in liver

on purine in the

broken-cell

nucleotides,

presence

and

the

of glucagon.

METHODS

o-enol-pyruvate I),

(trisodium

dithioerythritol

Radioactive

materials

from from

University

522

Bio-Rad, Holtzman of Utah

salt),

(DTE),

were

purchased

intact

cyciase

Prostaglandins

5OW-X4

from

of adenylate

(Type

be reproduced

used

has not

AND

phosph

have

on

concentrations

influence

action.

been

depend

AMP

also

of prostaglandin

to that

Michigan.

Corp.,

were

salt),

Chemical

Kalamazoo,

(250-350

II),

cyclic

can

studies

of this

MATERIALS Pyruvate

prostaglandins

stimulation

dependence

of prostaglandin

that

cyclase

we show

PGEl

site

catecholamines

increase

organs

prostaglandin

of the

adenylate

thus

and

in many

established

of the

assignment

and

action

it has been

(2,3).

of glucagon

cyclase

of hormone

and

accumulation

effects

adenylate

consequence

prostaglandins,

tions,

glycogenolytic

provided

by

were

Alumina Co. Vivarium

ATP

and

glucagon

Dr.

John

Pike,

from

New

obtained (neutral,

Mice, D

(disodium

activity rabbits,

were

I) from and

Vol.

55, No.

BIOCHEMICAL

2, 1973

A PROSTAGLdNOlN

AND

BIOPHYSICAL

El STIMULATION

0.5 I.0 PROSTAGLANDIN

6. GLUCAGON

2.0 El (pg/0.2ml)

IO.0

9

RESEARCH

COMMUNICATIONS

STIMULATION

f

e

I

I

4

Fig. 1 . Incubation and assay conditions are described in Methods. Protein conce trations varied from 150-300 pg per incubation sample. GTP, when present, equaled 10 -4,. Values given are the means of 4 measurements, glucagon; 8 measurements, PGE . B;clckets 1 indicate standard error of the mean (S .E .M .).

Rats barbital. SC issors,

and

mice

Livers

were

and

weight)

at

cheese

pended

in

and

was

1,000 cloth

which made

at was

Incubation. 7.5;

10 mM -

Mg’+;

then

1,200

0.5 0.75

mE rnz

ATP;

and

to 1 .5 mg/ml

containing

The 0.6

10 min.

added 632Pl-ATP

The

volume

as determined

its

DTE

1,200

liver

weight),

preparation

gm

of a teflon was

were

homogenized made

with

(per

strokes

Pellets

was

pheno-

minced

homogenate

15 min.

suspension

particulate

Six

The

with

10 volumes

(buffer).

x 9 for

rabbits

excised, in

tissue.

of 0.2 0.03

pyruvate

pyruvate (2-4

homogenizer

A second

theophylline;

with

blood,

and

filtered resus-

as above, in 5 volumes

used

of

in incubations

in buffer.

incubation

un

to remove

on original

pellets

mM -

dogs

1 mM -

at

as above. these

ether,

to homogenize

(based

centrifuged

7

(NH4)2S04,

saline

centrifuged

x g- for

diethyl

I ve h i e m

used

of buffer

phospho-enol-pyruvate;

22

a Potter-E

t-pm were

by suspending

0.9%

pH 7.5

and

10 volumes

centrifuged

buffer

with

with

with

- M Tris-HCI

turning

through

anesthetized

perfused

disrupted

of 0.01

pestle

were

x

lo6

by the

ml

contained:

mg

bovine

kinase;

cyclic

mM -

DTE,

0.25

CPM).

M em b rane

523

of

serum

1 mM -

kinase;

procedure

22 mM -

Lowry

protein et al.

Tris-HCI,

albumin; AMP;

added

9 mM -

5 mM with

KCI;

enzyme;

in incubations (4).

pH

was

Vol.

55, No.

BIOCHEMICAL

2,1973

Incubations

were

for

15 min

AND

BIOPHYSICAL

at 370 and

were

RESEARCH

terminated

by

COMMUNICATIONS

immersing

the

samples

n

in boiling cyclic

water AMP

for

Krishna of

After the

AMP

et al.

columns the

were

of [‘HI

cyclic

previously

under

the

Dowex

buffer

was

used

the alumina and

This

vial.

Scintillation

AMP

were

added

to monitor

solution (Bio-solv

ADP

so that to elute

the

during

this

cyclic

AMP

step

were

was

collected

the

measured 100 cpm

and (0.5

the

Dowex.

under

x

alumina.

Effluent

Dowex

columns

continued

an air

POPOP

a Packard

using

buffer,

with

in a scintillation

dimethyl

with

buffer).

columns

column

to dryness and

(elution

onto

The

alumina

PPO,

less than

flow

from

the

taken

x 4 cm

Alumina

was discarded.

from

to 0.5

4 ml elution

would

AMP

described

pH 7.6

with

effluent

of that

applied

was discarded.

8 ml of effluent

radioactivity

were

- M Tris-HCI

cyclic

and

ml)

washed

further

- Beckman,

of radioactivity

0.01

were

and

was warmed

was added,and

levels

ATP

of 0.25

with

columns

total

was a modification

volume

rinsed

of the The

solution H20)

column

elution

counting

chromatography (final

of the

buffer.

Blank

samples

most

placed

removed,

initial

Dowex,

containing

from

ml

amounts

the

8 ml of elution

0.2

The

entered

ml of elution

obtained

An

5OW-X4

sample

were

Four

assay.

(5).

Dowex

effluent

6 cm)

Tracer

recovery.

Cyclic by

3 min.

2-6

stream.

in toluene,

tri-carb

plus

spectrometer.

x TO6 cpm

of substrate.

in different

species

RESULTS Considerable noted;

variation

therefore

adenylate cyclase degree

prostaglandin

cyclase from

in the

was

these

El

of stimulation.

Data

responds In all

of guanosine-5’-triphosphate that

caused

by glucagon

Activation is also diesterase cluded of adeny

in Table has a negligible

that late

the

increases

ion, I.

of prostaglandins activation

given

stimulation

(GTP),

and

but

surpassed

which Under effect in cyclic

in Toble

of rat,

mouse,

I show

that

to PGE 1, although

cases,

alone

by fluoride given

(PGE,)

verified.

animals

action

by

stimulation

assay

on the AMP

PGEl by

accumulation indicated

cyc lase .

524

is some

PGEl

+ GTP

with

to cause

conditions

the

near

described,

cyclic

of cyclic in Table

I are

dog

liver

adenylate

variation

in the

upon

the

presence

is comparable

glucagon optimal

and

liver

is dependent

by stimulation

is considered the

there

rabbit,

has been

+ GTP enzyme nucleotide

to (Table

activity, phospho-

AMP.

Thus,

it is con-

due

to direct

stimulation

I).

Vol.

55, No.

BIOCHEMICAL

2, 1973

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Fig. 2. Incubation and assay conditions are described in Methods. Components were present as ‘ndicated at the following concentrations: Fluoride = 10 mM, glucagon = 10 -ifi hJ PGEl = 10 pg/ml, GTP= 10m4 M. Basal specific act;;-ity was 0.24 nmole cyclic AMP mg-l protein 15 min’l . Braxets indicate 2 S .E .M. for six measurements.

Fig 0 1A describes using

the

pg/ml

particulate

(1 .4 PM)

tions

of PGE,

and

the

Krishna

Fig.

1 B shows

same

with

with PGEl

lation optimal

are

studies summarized

caused

stimulation

(6),

and

Wolff

et al.

30 pM

PGEl

(7).

measured

with

1B shows

which

that

GTP

stimulates with

to determine

any

more 0.01

FM

pg/ml

shown

identical

data

for

than

.

membrane the

adenylate

conditions

1A was obtained.

+ GTP.

concentrations

Concentra-

concentration.

that

greater

PGEl

0.5

incubation

Fig.

gives

detail, with

of platelet

have

+ PGE,

of PGE,

2.5

using the

in more

occurred

PM)

optimal

observed

Thus, the

in Fig.

by glucagon

concentrations

(2 GTP). increase

being

(7.0

reported

preparations

+ GTP

pg/‘ml

as the

stimulation

1A and

stimulation

stimulation

glucagon

GTP

stimulation

than

Glucagon

is more

as compared

to

1 .41 pM

.

Further glucagon

PGEl

2.5

on

same

by

glucagon

effects

have

Threshold

with

the

its dependence

liver.

is stimulated

of Figures that

rat

gave

2 pM

particulate

but

and

stimulation

et al.

with

thyroid

Comparison

potent

optimal

from

glucagon

from

to 100 pg/ml

cyclase

cyclase

activation

fraction and

In comparison, adenylate

PGEl

the

activity

2.

1

hormone

measured

in the

the

However,

does

to that are

of PGEl

stimulation

inclusion

stimulation

stimulations presence

on the

E, (lOpg/ml)

+ GTP.

increases

individual

of PGE,

Prostaglandin

or glucagon of PGE

effect(s)

not

not

caused alter

the

of glucogon measured

additive,

in vitro

stimulation

of liver

adenylate

525

cyclase

.

by

PGEl

reported

glucagon

glucagon

DISCUSSION The

stimu-

with

with but

by

here

will

(1 .O)

0.086

0.051

0.722

Dog

Rabbit

(1 .O)

(1 .o>

(1 .O)

It- 0.036

0.168

Basal

Mouse

Rat

Animal

CYCLASE

I ACTIVITY

( 9.1)

3.321

0.601

0.516

( 4.6)

(11 .8)

( 6.0)

+ 0.349

1.535

Fluoride

(1 .06)

0.671

0.068

0.098

(0.93)

(1 .3)

(1 .l)

+ 0.026

0.179

PGE,

(4.1)

+ GTP

1 .558

0.251

0.148

(2.2)

(4.9)

(1 .7)

-r 0.088

0.688

PGE,

0.911

0.099

0.108

20.108

0.354

GTP

(1 .3)

(1.9)

(1.3)

(2.1)

2.616

0.447

0.453

20.324

1 .283

(3.6)

(8.8)

(5.3)

(7.6)

Glucagon + GTP

(4.4)

1 ,225

0.281

0.222

(1 .7)

(5.5)

(2.6)

+ 0.159

0 .745

G lucagon

-1 -1 Adenylate cyclase activity is expressed as nmole cyclic AMP biosynthesized mg protein 15 min . Fold increases over basal activity are given in parentheses. All determinations are in duplicate except for rat, which ore the means of 20 measurements on nine animals (2 S .E .M .). Preparation of particulate fractions used is described in Methods. Activity was measured within 60 min of enzyme preparations. The incubation volumes (0.2 ml) contained 150-300 pg (rat), 150 pg (mouse), 86 Pg (dog), and 80 Pg (rabbit) of protein ‘, Components were present as irnricated at the following concentrations: fluoride = 10 m&J glucagon = 10 5i, PGET = 10 pg/ml, GTP = 10 JvJ.

ADENYIATE

TABLE

Vol.

55, No.

2, 1973

establishes

the

Previous

BIOCHEMICAL

possibility

results

have

Examining caused

(8).

Although

the

Others

concluded

have

(10).

reported

between

apparent

in that

induction

of tyrosine Binding

prostaglandin required

The ,-elating

been

noted

any

direct

nucleotide

was

on hormone

others

(6,15),

the

ATP

concentrations

dependency

of

in situ

action

first

reported

lock

of thyroid

adenylate

the

of nucleotide

Prostaglandins

above

of PGEl

on

cyclase

(7).

also

antagonize

PGE,

in

GTP

direct

of

effect

(Fig.

IA)

PGE,

(15),

and

has been

has been

AMP

can

are

(12).

by purified

liver

data

plasma

of similar

with

be established. uncertainty hormone

effects

of this

not

concentrations by

in

in vitro

on GTP

by

requires shown), from

low the 0.1

to

10m4 - M ITP or XTP.

prostaglandin

caused

be

As reported

(results

ATP

cyclasewill

can

be replaced

increases

cells

to adenylate

observed.

studies

reported

liver

related

dependency

with

in rat

suppressed

rats

is another

et al.

GTP

527

liver

but

cyclic (9).

perfusion

more

Enhancement

is observed

cyclic

presented

glycogenolysis

In our

change

of an adenylate

liver.

tissues

for

in liver. release

not

fetal

(1 I),

Possibility

liver

on

medium.

specificity

not

has been

of hormone-stimulation

requirement

were

discrepancies,

by Rodbell

on GTP

does

in adrenalectomized

the

of PGE,

incubation

liver

of prostaglandins

in other

stimulation

Furthermore,

A similar

role

mediated

on cultured

membranes

of the

AMP

has a more

by

hydroxylase

supporting

stimulation

in the

results

effects

stimulation

to the

observation

PGE,

(14),

PGE,

caused

PGE,

binding

of PGE,

results

by GTP

specific

cyclic

of PGE,

in rat

detailed

by glucagon

In fight

dependency

these

responses

mM. -

and

of PGE,

data.

arylhydrocarbon

(13),

increased

of PGEl

plasma

injection

in phosphophosphorylase

that

production and

to liver

glycogenolysis.

intravenous

in terms

possibility

but

glucose

stimulate

interaction. before

with

perfusion

glucagon

of glucagon

has also

compatible

by our

aminotransferase

activation

membranes

2.0

both

increases

production,

increased

COMMUNICATIONS

in controlling

that

(I) and

the

that

glucose

Similarities

cyclase

(8),

role

reported

interpreted

is supported

Extone. or alter

are

adrenal

RESEARCH

possibility.

synthetase

were

BIOPHYSICAL

has some

et al.

changes

glycogenolysis

levels

this

observations

from

on hepatic

hormone

Curnow

of these

above

catecholamines

AMP

liver,

in glycogen Both

the

this

not suggested rat

decreases

activity

that

AND

by

stimulation

epinephrine

in fat

BIOCHEMICAL

Vol. 55, No. 2,1973

cells

(16)

and

inhibit

therefore

of interest

in

These

liver.

of PGE,

hormonally

induced

to examine

results

AND BIOPHYSICAL

effects

(Fig

a 2) show

cause

increases

responses of PGE,

that

RESEARCH COMMUNICATIONS

in several

other

tissues

on glucagon-stimulated

glucagon

stimulation

(17).

It was

adenylate

is unaffected

cyclase

by addition

. Prostaglandins

However,

studies

directly

using

stimulating

diestemse

intact

the

other

of adenylate

cyclase

prepamtions

(2O),

corpus

evidence

that

increasing in a number

ccl Is have

adenylate

. On

from

or decreases

cyclase hand,

or by

in vitro

thyroid luteum

not

(7), (21),

prostaglandins

in cyclic

determined

cyclic

experiments erythrocytes

and

in this

may

have

paper,

in many

prostaglandins AMP

shown

(18),platelets

be geneml

levels

whether

inhibiting

rat

AMP

liver. stimulators

act

degrading the

tissues. by

phospho-

direct

stimulation

(6,19),

cell

Therefore,

there

of adenylate

culture is cyclase

of tissues. ACKNOWLEDGMENTS

Utah

Partial Research

financial Fund.

We

assistance for this thank Mr. Kerry

research Welch

was received for technical

from the assistance.

University

of

REFERENCES 1. 2. 3. 4. 5. 6.

87: 9. 10. 11. 12. 13. 1’;:

2

Robison,

G.A., Butcher, R.W., and Sutherland, E.W. Cyclic AMP, Academic Press, New York, 1971 . Ramwell, P.W., and Shaw,J.E., Rec. Prog. Horm. Res., 26, 139 (1970). Kuehl, F.A., Humes, J.L., Cirillo, V.J., and Ham, E.A., Advan. Cyclic Nucleotide Res., 1, 493 (1972). Lowry, 0 .H . , Rosebrogli, N .J., Farr, A.L., and Randall, R.J., J. Biol. Chem., 193, 265 (1951). Krishna, G., WZss, B., and Brcdie, B.B., J. Pharmacol. Exp. Ther., 163, 379 (1968). Krishna, G., Yarwood, J .P., Barber, A. J . , and Jamieson, G .A., J. Biol . Chem., 247, 2253 (1972). Wolff, J., an=ok, G.H., J. Biol. Chem., 248, 350 (1973). F.Q., J. Biol. Chem., 247, 1892 (1972). Curnow, R .T . , and Nuttall, Exton, J.H., Robison, G.A., Sutherland, E.W., a&P&k, C.R., J. Biol. Chem., 246, 6166 (1971). Lemberg, A., WTfcinski, R., lzurieta, E.M., Halperin, H., Paglione, A.M., De Neuman, P., and Jauregui , H ., Biochim. Biophys. Acta, 248,198(1971). Gielan, J.E., and Nebert, D.W., J. Biol. Chem., 247, 7591 (19m Kaiita, Y., and Hayaishi, Q., Biochim . Biophys. Acta, 261, 281 (1972). Birnbaumer, L., and Pohl, S.L., J. Biol. Chem., 248, 20% (1973). Smigel, M., and Fleischer, S., Fed. Proc. Abstmcts, 32, No. 1298 (1973). Rodbell, M., Birnbaumer, L., Pohl, S.L., and Krans,H.M., J. Biol. Chem., 246, 1877 (1971). ButcheD .W., and Baird, C .E . , J. Biol. Chem., 243, 1713 (1968). Ramwell, P.W., and Rabinowitz, I., in Effects of Drus on Cellular Control Mechanisms (eds. B .R. Rabin and R .B. Freedman Park Press, 7 , University London, 1972 .

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BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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COMMUNICATIONS

6, 425 (1970). Res. Commun (1971).

.,

I,