Inhibitory action of adenosine 3′,5′-monophosphate on phosphatidylinositol turnover: Difference in tissue response

Vol. 104, No. 1, 1982 January 15, 1982

BIOCHEMICAL

INHIBITORY

OF ADENOSINE

ACTION INOSITOL

TURNOVER:

AND

BIOPHYSICAL

3';5'-MONOPHOSPHATE

DIFFERENCE

Departments

Toshikazu

COMMUNICATIONS Pages 105-112

ON PHOSPHATIDYL-

IN TISSUE

Kozo Kaibuchi,?' Yoshimi Takai, Shuji Kimura and Yasutomi Kobe University

RESEARCH

RESPONSE*

Yasuhiro Nishizuka

Ogawa,

of Biochemistry and RadioZogy School of Medicine, Kobe 650, Japan

Nakamura,

Akito

Tomomura

and Akira

Ichihara

Institute for EnzymeResearch, School of Medicine University of Tokushima, Tokushima 770, Japan Received

September

22,1981

There appear to be considerable differences among tissues in the inhibitory action of adenosine 3' ,5'-monophosphate (cyclic AMP) on phosphatidylinositol (PI) turnover induced by various extracellular signals. The present studies were on human peripheral lymphocytes and rat hepatocytes. In the lymphocyte system, cells are activated by phytohemagglutinin that induces PI turnover, and this PI turnover and cellular activation are profoundly blocked by dibutyryl cyclic AMP as well as by prostaglandin El which markedly increases cyclic in the hepatocyte system, glycogenolysis is enAMP. In contrast, hanced by a-agonists that induce PI turnover as well as by B-agonists and glucagon that increase cyclic AMP. In these cells the two classes of receptors appear to function independently, and PI turnover is not inhibited by cyclic AMP. SUMMARY:

In most for

the

One is

control related

tissues

there

of cellular to cyclic

appear

to be two major

activities AMP,L'

by various

and the

other

seems

receptor extracellular

mechanisms signals.

to be implicated

in

*/ This investigation was supported in part by research grants from the Scientific Research Fund of the Ministry of Education, Science and Culture, Japan (1979-1981), the Intractable Diseases Division, Public Health Bureau, the Ministry of Health and Welfare, Japan (1979-1981), a Grant-in-Aid of New Drug Development from the Ministry of Health and Welfare, Japan (1979-1981), and the Yamanouchi Foundation for Research on Metabolic Disorders (1977-1981). §/ To whomcorrespondence should be addressed at the Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan. L/ The abbreviations used are: cyclic AMP, adenosine 3',5'-monophosphate; PI, phosphatidylinositol; PHA, phytohemagglutinin; PGEl, prostaglandin El; DBcAMP, dibutyryl cyclic AMP.

0006-291X/82/010105-08$01.00/0 105

Copyright 0 I982 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 104, No. 1, 1982

actions

Ca2+

of

the

lipid

(for

latter

review,

class

turnover

ly

3).

divided

other

platelets

PI turnover

promote that

although in the to

other

interact

with

in hepatic cyclic

type

(4,5).

It

through

activation

analogous

activated,

AMP usually

antagonize

is

but

function

(6-8).

we examined

The results

lymphocytes dependent

but

proposed

that

species

protein to

In the present whether

cyclic cyclic

as protein

kinase

using

AMP interacts

AMP-dependent kinases

that

recently

Ca2+ -activated,

and cyclic

and equally

roles

kinase.

kinase,

studies

appear

In an

may be coupled

AMP inhibits

in hepatocytes.

kinase

tentatively

protein

do not

PI turnover

protein

protein

In contrast

For instance,

AMP plays

PI turnover

of

unclear.

glycogenolysis

cyclic

AMP-dependent

showed that

not

that

the

such activation,

induce

enhance

induce

whereas

of receptors

which

established

that

independently.

respectively,

of another

remains

two classes

of cyclic

tissues

hepatocytes,

referred

functions,

the

review,

can be rough-

the receptors

antagonism

(for

such as lymphocytes,

cellular

phospholipid-dependent

various

over.

system

has been well

the activation

of

in acetyl-

neurotrans-

response

a- and B-agonists,

manner it

by hormones,

tissue

mode of

phospho-

shown by many

this

this

each other

(2)

substances

cyclic

AMP accumulation,

and Hokin

and subsequently

activated

stimulation This

active

activation

of

tissue,

by Hokin

In most systems,

the mechanism of

the

PI turnover.

and smooth muscle,

produce

COMMUNICATIONS

biologically

two types:

leukocytes,

receptors

tissues

RESEARCH

In general,

induces

glands

Presumably, into

1).

recognized

in various

and various

see Ref.

receptors

excretory

investigators

BIOPHYSICAL

see Ref.

was first

choline-sensitive

mitters

of

AND

to 2+ is Ca found

in

lymphocytes with

and

PI turn-

PI turnover

in

phospholipidprotein

kinase

are

C and A, respectively.

EXPERIMENTAL PROCEDURES CellsWashed human peripheral lymphocytes were prepared by the method of Bijyum (9). The preparation consisted of 90-95% lymphocytes and 5-10% granulocytes and/or monocytes. The lymphocytes were

106

Vol. 104, No. 1. 1982

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

suspended in Eagle's medium at a concentration of 1 x 106 cells/ml, and maintained for 20 hr at 37“C under 5% CO2 in air. Parenchymal hepatocytes were isolated from adult rats as described earlier (10). The cells (5 x 105 cells/ml) were plated in Williams' medium E supplemented with 5% calf serum and 10 FM dexamethasone, and cultured for 7 hr at 37OC under 5% CO2, 45% Orand 50% N2. These cells accumulated glycogen and maintained various hepatic functions, including responses to hormones such as glucagon, epinephrine and insulin, urea synthesis, albumin synthesis and enzyme induction as described (11). Materials and Chemicals -The materials and chemicals used for preparation of i$iiphocytes and hepatocytes were obtained as described earlier (10,12). PHA and PGEl were products of Difco Laboratories and Ono Pharmaceutical Co., respectively. DBcAMP, glucagon and phenylephrine were purchased from Sigma. Epinephrine and isoproterenol were obtained from Nakarai Chemicals. 32Pi and 13Hlthymidine (5 Ci/mmol) were from the Radiochemical Centre. Other chemicals were obtained from commercial sources. Procedures and DeterminationsPI turnover was usually assayed by measuring theincorporation of 32P into PI as described originally by Hokin and Hokin (2) and later established by many investigators (3). The radioactive phospholipids were extracted directly with chloroform/ methanol (1:2) by the method of Bligh and Dyer (13), isolated by thin layer chromatography on Silica Gel H (E. Merck) plates, and determined as described earlier (14). DNA synthesis was assayed by measuring the incorporation of [3H]thymidine into acid-precipitable materials as described by Smith et al. (15). Glucose output from hepatocytes was assayed enzymatically with glucose oxidase as described (16). Cyclic AMP was determined by radioimmunoassay by the method of Steiner et al. The radioOther conditions are specified in each experiment. (17). activity of 32P- and 3H-samples was determined using a Packard TriCarb liquid scintillation spectrometer, Model 3320.

RESULTS AND DISCUSSION

Polyclonal in

plant

lymphocytes

employed was greatly labelling ment of

(18,19).

The results studies

increased

by PHA, but

was observed labelling

radioactivity

diversity

such as PHA induce

in the present

of PHA, and the

small,

mitogens

reaction

the

in different

of phosphatidic

that

other

the

that

acid

continued in

incorporation

tissues

linearly

of this

(data

in lymphocytes

not

32P into

for

at least

acid

fraction

of

the addition 60 min.

phospholipid

shown).

PI

The enhance-

after

was presumably

107

of

the cells

no enhancement

immediately

pattern

with

phospholipids.

the phosphatidic

labelling

PI turnover

I confirm

practically

major

of PI was observed

recovered

although

with

in Table

rapid

The poor

The

was very showed great labelling

due to its

rapid

Vol. 104, No. 1, 1982

BIOCHEMICAL

AND

BIOPHYSICAL

Table

RESEARCH

COMMUNICATIONS

I

32 PHA-induced enhancement of P-incorporation into PI in human peripheraz lymphocytes

Phospholipid

Without

Phosphatidylinositol Phosphatidic

PHA

With

690 acid

PHA

4,060

400

500

Phosphatidylserine

1,950

1,730

Phosphatidylethanolamine

5,000

5,700

Phosphatidylcholine

3,400

3,600

Human peripheral lymphocytes were suspended in phosphate-fr e KrebsRinger bicarbonate buffer at pH 7.5 at a concentration of 1 x 10 & cells/ml. The suspension was incubated with carrier-free 3zPi (30 uCi/ml) for 1 hr at 37”C, and then stimulated by PHA (10 pg/l x 106 cells) for 30 min. Phospholipids were extracted and isolated by thin layer chromatography using a soIvent system of chloroform-methanol-acetic acid-water (50:30:8:4). For separating PI and phosphatidylserine an additional solvent system of methylacetate-n-propanol-chloroform-methanol-0.25% aqueous KC1 (25:25:25:10:9) was employed. The numbers indicate the radioactivity (counts per min) incorporated into each phospholipid.

conversion

to PI by way of CDP-diacylglycerol

DNA synthesis tion

into

was enhanced

the acid-precipitable

challenge.

Although

of PI turnover equally cyclic

the

inhibited

causal

inhibited

of

progressively

was inversely

effect

is

not

II.

of

of

by increasing

lymphocytes,

entities

toxin,

Agents

increase<

per se had no effect. that into

amounts of PGEl,

DBcAMP.

PHA

were

markedly

1 indicate

AMP level.

incorpora-

the enhancement

which

[3H]thymidine

to the cyclic by

both

Sodium butyrate

such as PGEl and cholera

alterations

clear,

Later,

72 hr after

between

as by PGEl,

PI and that

related

i3H]thymidine

was maximal

shown in Fig.

of PGEl was reproduced

AMP level, induced

results

32 P into

of

relationship

by DBcAMP as well

AMP as shown in Table

corporation

rate

fraction

and DNA synthesis

The more quantitative

tion

and the

(PI turnover).

both

DNA were

and this

This increasing

in-

inhibi-

inhibitory the cyclic

are known to prevent PHA2+ such as enhancement of Ca trans-

108

BIOCHEMICAL

Vol. 104, No. 1, 1982

AND

BIOPHYSICAL

Table Inhibition

32 enhancement of P-incorporation into DNA in lymphocytes 3 [ H]Thymidineincorporation into DNA

32 P-Incorporation into PI

Cyclic AMP formation

(cpm)

(cpm) None

COMMUNICATIONS

II

by PGEl and DBcAMP of PHA-induced into PI and [3H]thymidine-incorporation

Addition

RESEARCH

(pmol/l

x 10 6 cells)

830

420

0.2

PM (10 ug/ml) -5 PGEl (1 x 10 M) -3 DBcAMP (1 x 10 -M)

3,630

1,710

0.2

820

430

2.2

790

510

-

PHA + PGEl

1,450

670

3.0

PHA + DBcAMP

1,250

560

-

32 P-Incorporation into PI was assayed as described in the legend to Table I except that PGEl and DBcAMP were added as indicated during cell activation. For the assay of DNA synthesis, non-radioactive lymphocytes (1 x lo6 cells/ml) suspended in Eagle's medium were stimulated by PHA for 72 hr in the presence and absence of either PGEl or DBcAMP as indicated, and then incubated with [3H] thymidine (1 uCi/ml) for 2 hr at 37°C. Cyclic AMP was measured after incubation of the cells for 10 min at 37'C with and without PHA and PGEl as indicated.

port,

glucose

DNA syntheses inhibitory

protein

(for

action

PI turnover

see Ref.

of cyclic

AMP is

that

of

was also

by various extracellular

signals

in human platelets

stimulated

leukocytes

stimulated

activation

were

blocked

elevated

the

markedly

The above findings

clear,

the

activation C.

observed

signals

by thrortbin

raise

inhibition types

cyclic

question

109

not

may be

by another

AMP.

For instance,

polymorphonuclear

PI turnover

(data

by cyclic which

inhibited

and rat

this

a multifunctional

and cellular

by DBcAMP as well

AMP level the

cell

but

increase

concurrently

and RNA and

AMP may block of

Similar

by a chemoattractant,

cyclic

cyclic

in other

that

syntheses

The mechanism of

not

kinase

extracellular

acid

20).

counteract

is protein

AMP of PI turnover

group

and fatty

review,

and thereby

kinase

activated

sterol

transport,

as by PGEl which

shown).

of whether

cyclic

AMP inhi-

Vol. 104, No. 1, 1982

BIOCHEMICAL

I

0

AND

1

lo-

'

BIOPHYSICAL

I

10+

I

lo-'

0

1.

Effects

enhancement DNA in DNA, in

of

lymphocytes. and

the

were

added

tion

into

various

AMP were to

Table

as indicated. PI;

concentrations

measured II

except A,

(o-----o),

I

lo- 4

1O-3

DBcAMP (Ml

32 P-incorporation into 32 P-Incorporation

cyclic legend

of

COMMUNICATIONS

I

lo-'

PGEl CM) Fig.

RESEARCH

with

of

PI

and

into

under

PGEl

various

PGEl;

DBcAMP

on PHA-induced

[3H]thymidine-incorporation PI,

into

[3H]thymidine-incorporation

conditions

that

and

similar

to

concentrations

B, with

of

DBcAMP.

[3H]thymidine-incorporation

(-----@I DNA;

into

into

those

described

PGEl

and DBcAMP 32 P- incorporaI (A---4), cyclic

AMP.

bits

PI turnover

two receptors

function

established through

in the other

that

a cyclic

a-agonists,

glycogenolysis

vasopressin without

level

activation

a-agonist

but

in Table

was not

Preliminary

analysis

is

enhanced

pathway.

inhibited indicated

showed

is

kinase

by B-agonist, insulin

over.

110

also

provoke

PI

Under these

that

hepatocytes

increase

that

where the

by B-agonists

It II

of protein

not by B-agonist.

PI turnover

In isolated

any detectable

III

such as liver

system

and angiotensin

as glycogenolysis

ments summarized

of

independently.

AMP-dependent

and without

type

well

known that

PI turnover in

the

conditions glucagon not

as well

cyclic

AMP

The experiwas

turnover

seems

and glucagon

A (3,4).

did

it

enhanced

this

enhanced

or DBcAMP. inhibit

by

PI turn-

Vol. 104, No. 1, 1982

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Table III Effects of cycZic enhance4nent of

AMP-elevating 32P-incorporatiun

compounds on a-agonist-induced into PI in rat hepatocytes

32P-incorporation into PI

Addition

Glucose output

( 91 None Epinephrine

(1 x 10 -' -M)

Cyclic AMP formation (pmol/mg of protein)

(Whrhg of protein)

5,850

46

2.5

12,260

100

69.9 8.4

Phenylephrine

(1 x 10 -' -M)

10,820

83

Isoproterenol

(1 x 10 -' M) -

5,430

102

5,390

95

149

5,380

110

-

10,180

104

83

9,680

105

138

10,940

110

Glucagon

(1 x lo-'

-M)

DBcAMP (1 x 10 -4 M) _ Phenylephrine

+ Isoproterenol

Phenylephrine

+ Glucagon

Phenylephrine

+ DBcAMP

79.9

Cultured rat hepatocytes (1 x lo6 cells/35 mm dish) were incubated with carrierfree 32Pi (50 uCi/ml) for 1 hr at 37'C, and then stimulated for 30 min at 3°C by 3*P-Incorporation into PI was measured as described various compounds as indicated. in the legend to Table I. Glucose output was assayedafter incubation of the cells for 30 min at 37°C with various compounds as indicated in glucose-free Hanks-Hepes buffer at pH 7.2. Cyclic AMP was measured after incubation of the cells for 10 min at 3°C with various compounds as indicated.

Protein including

all

kinase

the cell

with

human platelets

this

protein

by the protein

C is widely

kinase

types

receptor-linked

activated

during

between

not yet

been clarified

tractive

to suggest

that

in

the

receptor-linked

activation

lic

AMP, presumably

through

above

of PI, platelet

A series

strongly

which

and is

of protein action

111

derived

involved

(21,22).

it

in

Although

response

and hepatocytes, of system

that

is

directly

and the cellular

the type

of studies

suggested

activation

lymphocytes

the

(8).

by diacylglycerol,

PI turnover for

in mammalian tissues

by thrombin

hydrolysis

phosphorylation

the relationship

mentioned

stimulated is

distributed

has

seems

at-

such as lymphocytes,

kinase of protein

C is blocked kinase

by cyc-

A, whereas

Vol. 104, No. 1, 1982

in the

type

independently

of

BIOCHEMICAL

BIOPHYSICAL

system such as liver, in their

ties.

The inhibition

pear

to be due simply

the molecular

AND

basis

specific

protein

roles

of PI turnover

this

difference

kinases

by protein

in

COMMUNICATIONS

C and A function

in controlling

to the phosphorylation of

RESEARCH

cellular

kinase

activi-

A does not

of phospholipase tissue

response

are grateful

to Mrs.

ap-

C, and

remains

unexplored. The authors

Acknowledgementand Miss K. Yamasaki

for

their

skillful

secretarial

S. Nishiyama

assistance.

REFERENCES 1. Berridge, M.J. (1975) Adv. Cyclic Nucleotide Res. 5, l-98 2. Hokin, L.E. and Hokin,xR. (1955) Biochim. Bzhys. Acta 18, 102110 3. Michell, R.H. (1975) Biochim. Biophys. Acta 415, 81-147 4. Kirk, C.J., Verrinder, T.R. and Hems, D.A. (1977) FEBS K. 83, 267-271 5. Murad, F., Chi, Y.M., Rail, T.W. and Sutherland, E.W. (1962) J. Biol. Chem. 237, 1233-1238 6. Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695 Y., Kishimoto,A., Kikkawa, U., Mori, T. and Nishizuka, Y. 7. Takai, (1979) Biochem. Biophys. Res. Commun. 91, 1218-1224 8. Minakuchi, R., Takai, Y-,x, B. and Nishizuka, Y. (1981) -J. Biochei 89, 1651-1654 9. zyum, A. (1968) Stand. J. Clin. Lab. Invest. 21 (suppl. 97), 77-89 and Ichihara,A. (1978) -J. Biochem 10. Tanaka, K., Sato, M., Toi%ta,. 84, 937-946 11. Ichihara, A., Nakamura, T., Tanaka, K., Tomita, Y., Aoyama, K., Kato, S. and Shinno, H. (1980) e. N.Y. Acad. Sci. 349, 77-84 12. Ogawa, Y., Takai, Y., Kawahara, Y., giiiiura, S. and Nishizuka, Y. (1981) J. Immunol. in press 13. Bligh, E.G. and Dyer, W.J. (1959) -Can. J. Biochem. Physiol. 37, 911-917 14. Takai, Y., Kaibuchi, K., Matsubara, T. and Nishizuka, Y. (1981) Biochem. Biophys. Res. Commun. 101, 61-67 -15. Smith, J.W., Steiner, A.L. and Parker, C.W. (1971) 2. Clin. Invest. -50. 442-448 16. !&omura, A., Nakamura, T. and Ichihara, A. (1980) Biochem. Biophys Res. Commun. 9J, 1276-1282 17. -. Steiner,A.L., Pagliara, A.S., Chase, L.R. and Kipnis, D.M. (1972) J. w. Chem. 247, 1114-1120 18. Fischer, D.B. and Mueller, G.G. (1968) E. Natl. Acad. Sci USA -* -* 60. 1396-1402 19. gsuzawa, Y., Osawa, T., Inoue, K. and Nojima, S. (1973) Biochim. Biophys. Acta 326, 339-344 (1980) Research- Monographs -in 20. Hume, D.A. and Weidemann, M.J. Immunology Vo1.2, Elsevier, Amsterdam -21. Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K. and Nishizuka, Y. (1980) J. Biochem. 88, 913-916 22. Kawahara, Y., TakaiFY., Minakuchi, R., Sano, K. and Nishizuka, Y. (1980) Biochem. Biophys. Res. Commun. 97, 309-317 --

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