Modulation of cyclic AMP accumulation in GH3 cells by a phorbol ester and thyroliberin

Vol.

129,

June

28,

No.

BIOCHEMICAL

3, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

898-903

Pages

1985

MODULATION OF CYCLIC AMP ACCUMULATION IN GH3 CELLS BY A PHORBOL ESTER AND THYROLIBERIN LAWRENCE A. QUILLIAM, Department University Received

May 20,

PAULINE R.M. DOBSON, and BARRY L. BROWN

of Human Metabolism & Clinical of Sheffield Medical School, Sheffield, SlO 2RX, UK

Biochemistry, Beech Hill Road,

1985

4~ phorbol-12,13-dibutyrate (PDBu) stimulated cyclic AMP accumulation in GH~ pituitary tumour cells in the presence of isobutylmethylxanthine. This effect persisted after preincubation of cells with cholera or pertussis toxins. In contrast, vasoactive intestinal polypeptide (VIP)-stimulated cyclic AMP accumulation was inhibited by PDBu in a dose dependent fashion (IC50=5.1nM). Thyroliberin (TRH) had a similar, but non-additive, stimulatory effect on cyclic AMP accumulation with PDBu, however it did not inhibit VIP stimulation. These results suggest that TRH may stimulate cyclic AMP accumulation through protein kinase C and that stimulation of adenylate cyclase by PDBu and TRH may occur distal to the guanine nucleotide binding regulatory proteins, N, and Ni. 0 1985 Academic Press, Inc.

The effects

on cellular

activation kinase

C (5,6).

via

AMP production

.

stimul

are

normally

appear

dependent

protein

activated

(9)

been shown

however,

whether

there

have been several

stion

of adenylate

(see

reports

cyclase

and

is

to be mediated kinase,

7,S for

tumour by

to produce this

of profound

TRH via

by phorbol

esters

is

stimulated

increase

action

is

in cyclic not known.

of receptor(13,14,15).

'Abbreviations used: HDTA, ethylenediaminetetraacetic acid; Hepes, N-2hydroxyethylpiperazine-N'-2-ethane sulphonic acid; IBMX, 3-isobutyl-lmethylxanthine; Ns, Ni, stimulatory, and inhibitory guanine nucleotide binding regulatory proteins of adenylate cyclase respectively; 4cPd2, 4sphorbol-12,13-didecanoate; 4BP, 4B-phorbol; PDBu, 4B-phorbol-12,13-dibutyrate; TRH, thyroliberin (thyrotropin releasing hormone); VIP, vasoactive intestinal polypeptide. 0006-291X/85

Copyright All rights

$1.50 0 I985 by Academic Press, Inc. of reproduction in any form reserved.

898

by

phosphoinositide

a small

of modulation

a product

reviews).

cells

a direct

by

protein

by diacylglycerol,

pituitary

cyclase

to have a number

actions

breakdown

from GH3 rat

TRH has also

known

and their

and phospholipid

of adenylate

(11,12),

Recently,

(l-4)

phosphoinositide

activation (10)

esters

enzyme is

secretion

hydrolysis

mediated

This

induced

Prolactin

phorbol

function

of the calcium

of agonist

VIP'

promoting

tumour

Using

Vol.

129,

No. 3, 1985

bacterial

toxins

BIOCHEMICAL

we have

PDBu and TF!H on cyclic been presented

AND

therefore

tried

AMP metabolism

in a preliminary

BIOPHYSICAL

to identify in GH3

form

RESEARCH

COMMUNICATIONS

the site(s)

of action

Some of these

Cells.

data

of

have

(16).

MATERIALS

AND METHODS

Fetal. calf serum, trypsin/EDTA, penicillin and streptomycin were from Gibco-Europe, Paisley, Strathyclyde, UK. Hams's FlO and Dulbecco's modified Eagle's medium and GH3 cells were from Flow Laboratories, Irvine, Ayrshire, UK. Bovine serum albumin (fraction V), IBMX, cholera toxin and the phorbol esters were from Sigma Chemical Co., Poole, Dorset, UK. Pertussis toxin was a kind gift from Drs A. Robinson and L.I. Irons, Pathogenic Microbes Laboratory, Porton Down, UK. VIP was obtained from Peninsula Laboratories Europe, St Helens, Merseyside, UK. TRH was from Calbiochem-Behring, Cambridge UK. Adenosine :3'5' -cylic monophosphoric acid 2'-O- succinyl-tyrosine [ 115 Ilmethylester was from Amersham International, Amersham, Buckinghamshire, UK. All other chemicals were from BDH Chemicals Poole, Dorset, UK. Phorbol esters were dissolved in dimethylsulphoxide at a stock concentration of 2mM and stored in the dark at -2OOC. The concentration of dimethylsulphoxide in the incubations did not exceed 0.025% and had no effect on cyclic AMP accumulation. Monolayers of GH3 cells were routinely grown in Ham's FlO medium supplemented with 10% fetal calf serum and penicillin/streptomycin (100 units/ml). The cells were plated at a density of lo5 cells per well in culture meldium (lml) in 24 well multiwell dishes, and the experiments were performed 3 days later. The experiments were conducted in Dulbecco's modified Eagle's memdium containing Hepes (2OmM) and supplemented with 0.5% (w/v) bovine serum albumin (DMEM) at 37OC. Cells were washed twice with DMEM and then preincubated for 20 minutes with IBMX (1mM) in the presence or absence of phorbol esters or TRH. In experiments with VIP, the cells were incubated for a further 5 mins with the peptide. In experiments where bacterial toxins were employed, cells were preincubated for 24 hours with the pertussis toxin (50 ng.ml-') or for 2 hours with cholera toxin (1nM). The medium was removed and replaced by a mixture containing TRH or phorbol esters and IBMX for a further 20 or 30 minutes. Incubations were terminated by aspiration of the medium and the Cyclic AMP was extracted addition of ethanol (65% v/v; 4OC) to the monolayers. from the cell layer as described previously (17) and measured by radioimmunoassay (18). Significances were assessed by analysis of variance. RESULTS AND DISCUSSION As previously accumulation maximal

of these

The non-tumour didecanoate

both

(Table agents

AMP accumulation

(Table

promoting

the above

and TRH were

exerting

a similar suggesting

la),

esters,

but,

study,

cyclic

AMP

we also

found

non-additive

a common mechanism

4&phorbol

that

increase

in

of action.

and 4a-phorbol-12,13-

effect.

experiments

their

In the present

la).

phorbol

phosphodiesterase

PDBu and TRH stimulated

produced

had no significant

Since nucleotide

(ll),

in GH3 cells

doses

cyclic

shown

were

inhibitor, effects

performed

in the presence

IBMX (1mM) it on cyclic

of the cyclic

seemed likely

AMP metabolism

through

that

PDBu

activation

Vol.

129,

BIOCHEMICAL

No. 3, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

TABLE 1 me effect of phorbcl esters (5OOnBl) and TRH (2OOnH) on cyclic AW accumulation in CH3 calla under varioua conditions at 37% in the presence of IBMX (l&4) Addition

a)

IBMX

b)

Cholera

cyclic

15.35

-+

2.21

PDBu

24.77

-+

2.84*

4Bp

15.25

-+ 3.08

W'd,

16.63

+

1.96

TRH

23.06

-+

2.46*

PDBu & TRH

25.39

-+

3.oa*

VIP

77.28

-+

2.57*

(10nl4)

toxin

c)

Pertussis

toxin

AMP (pmol/well)

98.1

-+

6.58

18.31

-+

1.07

174.68

+

8.84*

29.54

-+

1.37*

103.62

-+

1.58

17.92

-+

1.14

143.76

-+

11.71*

24.59

-+

2.61*

107.0

-+

5.39

93.58

-+

4.76*

In 'a' cells were incubated for 20 minutes with IBMX + agents. In 'b', cells were preincubated for 2 hours with cholera toxin (1nM) and-IBMX prior to addition of agents for a further 30 minutes. Cells in 'c' were preincubated for 24 hours with pertussis toxin (5Ong.e1-'1 prior to addition of agents for 20 minutes. Cyclic AMP in the cell layer was measured by radioimmunoassay. Triplicate wells were used for experiment 'a', quadruplicate wells for 'b' + 'c'. Results representative of at least 3 experiments. *p
of adenylate cyclase which

cyclase

activation couples

studied

stimulatory

with

hour

able

to further

(20).

We therefore

accumulation inactivates

after Ni

toxin

(results

Ni can lead

by ADP-ribosylation

AMP accumulation

after

unit

(C).

activates Table

cyclic

(InM),

protein, We therefore

after

pre-

adenylate

lb that,

cyclase

unlike

AMP accumulation

a dose which

Ns,

VIP, after

produced

by

PDBu a 2

maximal

not shown). guanine

nucleotide

to enhancement

studied

pre-incubation

from

Adenylate

binding

AMP accumulation

stimulate

cholera

nucleotide

permanently

is clear

of the inhibitory

cyclase,

cyclase

It

AMP degradation.

to the catalytic

which

(19).

AMP accumulation

adenylate

on the guanine

toxin,

with

Inactivation

cyclic

N,

pre-incubation

cyclic

cyclic

of PDBu and TRH on cyclic

of both

inhibiting

receptors

cholera

ADP-ribosylation and TRH were

than

is dependent

the effect

incubation

rather

of activation

the effects of cells (19).

pre-incubation

900

binding

of

of adenylate

of PDBu and TRH on cyclic

with Both

protein

pertussis agonists

of cells

toxin, still

with

AMP

which stimulated

pertussis

toxin

(50

Vol.

129,

No. 3, 1985

BIOCHEMICAL

AND

O1o+

0:1

BIOPHYSICAL

i

io

PDEU

Figure

RESEARCH

loo

COMMUNICATIONS

1000

I-M

1

The eff&t presence

of of

PDBu on VIP IBMX (1mM)

(1OnM)

stimulated

cyclic

AMP

accumulation

in

the

Cells were preincubated for 20 mins with IBMX and PDBu. The medium was changed to a mixture containing VIP and the reaction was stopped after 5 mins by the removal of the medium and addition of ice-cold 65% (v/v) ethanol. Cyclic AMP in the cell layer was measured by radioimmunoassay. Representative of 3 experiments

performed

A = IBMX alone

ng.ml-')

.

in triplicate. = IBMX plus

24 hours

for

(Table

lc),

ADP-ribosylate

Ni in GH3 cells

their

It

action.

unit

therefore

(C) Iof adenylate In addition

inhibition tumour it

cyclase

phorbol

stimulation

of adenylate

It

is

therefore

AMP accumulation VIP receptor. attenuation

is

mediated

(10)

and produces

phorbol

esters

in GH cells

action

of VIP.

Similarly,

(221, phorbol

2 1.3nM;

esters

by phosphorylation

via

i.e.

with

TRH did

phosphorylation

it

clear

esters 901

but

not

S-adrenerqic receptor

why it

cyclic

phosphorylation

protein

not

Recently,

2).

of VIP-stimulated

a similar is

This

inhibit

TRH stimulates

2).

PDBU resulted

1).

(Table

Since

(Table

N, and C.

of the Badrenerqic

PDBu inhibition

preincubation

catalytic

mean + SD, n= 3) and the non effect

manner

on the

With

(Fig

and isoproterenol

a similar

for

between

of cells

AMP accumulation

a similar

of the VIP response

diacylqlycerol

pre-incubation

that in

coupling

had no significant

phorbol

not necessary

PDBu and TRH act

promote

(IC5D=5.1

possible

shown to maximally

Ni is

that,

cyclic

cyclase

In contrast,

suggesting

effect,

esters

that

SD).

previously

or possibly

was dose dependent promoting

+

treatment

seems likely

of VIP stimulated

has been reported

(15).

a (21),

to the above

in attenuation

(mean VIP

failed

TRH can inhibit

not

of the lead

to

the release pattern to inhibit epidermal

of to the

Vol.

129,

BIOCHEMICAL

No. 3, 1985

TABLE 2 The effect on

BIOPHYSICAL

of phorbol ester (5OOnM) cyclic MP accluulation

stimulated

VIP

AND

and TFM (2OOnM) in the presence

Cyclic

Treatment

RESEARCH

IBMX

-+

1.08

+ VIP

64.37

-+

2.06

PDBu + VIP

33.45

-+

1.92’

61 .63

+

6.08

4aPd2 + VIP

62.12

-t

0.90

TRH

66.42

-+

2.63

+ VIP

48P

+ VIP

preincubation of IBUX(teH)

AMP (pmol/well)

14.04

IBMX

COMMUNICATIONS

of VIP (IOnM) Cells were incubated for 20 minutes with agents, prior to addition Cyclic AMP in the cell layer was measured by for a further 5 minutes. radioimmunoassay. Results are the mean -t SD of triplicate wells and are representative of 5 experiments. *p
growth

factor

attenuation protein

binding

of VIP stimulated kinase

cyclic

(IC50

The data accumulation

in may

evidence

study

suggests

dependent

(26)

have demonstrated

upon guanine

This

why stimulation

receptor

binding

persisted

after

some

preincubation

controversy

and membrane phosphoinositide

toxin

stimulation over preparations hydrolysis

be

TRH

(28,291.

can

agonist

the stimulation

or cholera

and subsequent 902

in intact

activation

C.

with

toxin

TRH

cells,

possibly

of protein

in

this

C in GH3

cyclase

for,

that

AMP accumulation

of phospholipase

The requirement

et al

of phospholipase

with

adenylate

is

suggesting

has been associated

pertussis

AMP

induced

cyclic

stimulte

cells

Verghese

in leukocytes,

AMP production

cyclic cyclase.

in broken

TRH stimulated

regulators

of cyclic

whether

in

the

of adenylate

can inhibit

of GTPase activity

to

cyclic

stimulated

In addition,

hydrolysis

since

VIP)

hydrolysis

(24,25).

of cells

Ni and N, are unlikely Unlike

phosphoinositide

However,

(27).

in stimulating

to the N units

be involved

may

of

a common mode of action.

TRH (unlike

distal

bisphosphate

protein

exlain

that

pertussis

Ni or a similar

PDBu causes

independently

was equipotent

nucleotides

that

that

mean + SD n= 3) and in inhibiting

suggest

that

phosphatidylinositol-4,5-

cells.

it

suggesting

at a point

occur

also

study,

since

= 5.1 -+ 1.3nM), this

The possibility

AMP accumulation

(ED5C = 5.2 -+ 0.3nM;

VIP response

may

(23).

C seems unlikely

AMP accumulation

Recent

to GH cells

there

in broken

is cell

GTP dependent, kinase

C (an

Vol.

129,

enzyme cyclase

No.

largely

3, 1985

present

stimulation

modulation

BIOCHEMICAL

in the may explain

of adenylate

cyclase

AND

cytosol

BIOPHYSICAL

of unstimulated

why it

has been difficult

in membrane

RESEARCH

cells)

COMMUNICATIONS

prior

to adenylate

to observe

TRH

preparations.

ACKNOWLEDGEMENTS LAQ is the recipient of a studentship from the Isle of Man Board These studies were supported, in part, by grants from the Medical Council and the Yorkshire Cancer Research Campaign.

of Education. Research

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Litosch, I., Wallis, C. & Fain, J.N. Biochem Sot Transactions (in press). Cockcroft, S., Gomperts, B.D. (1985) Nature (London) 314, 534-536. Verghese, M.W., Smith, C.D., Snyderman, R. (1985) Biochem Biophys Res Commun. 127, 450-457. Hink:le, P.M. & Phillips, W.J. (1984) Proc.Nat.Acad.Sci. USA 2, 6183-6187. Hink:le P.M. C Tashjian, A.H. Jr. (1977) Endocrinology 100, 934-944. Gautvik, K.M., Gordeladze, J.O., Jahnsen, T., Haug, E. & Hansson, V. (1983) J Biol Chem. 258, 10304-10311. 903