Vol.

167,

March

No.

30,

BIOCHEMICAL

3, 1990

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1990

982-992

CYCLIC GMP STIMULATES INOSITOL PHOSPHATE PRODUCTION IN CULTURED PITUITARY CELLS: POSSIBLE IMPLICATION TO SIGNAL TRANSDUCTION Zvi Department of Sciences, Received

January

SUMMARY: cyclic rat

of

cells

nucleotide

myo-[2-3

results

to

the

via

effect the

enhanced

stimulate

is

formation

hormones

Several

1,2-diacylglycerol of

C, respectively platelets,

the

neutrophils,

0 1990 by Academic Press, Inc. in any form reserved.

of reproduction

inositol

lymphocytes)

982

via Ca2+-

turnover

also

then

serve needed

Press, Inc.

operate

bifurcating

via

internal

enhanced

pathway

1,4,5-trisphosphate

several

which

Many

might

provides (IP3)

and

Ca2+ mobilization

Ca2+/phospholipid-dependent In

and

phosphoinositides

ligands

for

which

(IP3)

nucleotide

The

the

1-monophosphatase,

Q1990Academic

required

(l-4).

Li+,

of

of

0006-291x/90 $1.50 Copyright All rights

of

of

as that

hydrolysis

inositol (DG)

effect

phosphoinositide

C activation.

messengers

effect

as well1

cyclic

(1).

[3H]-myo-

1,4,5-trisphosphate

via The

of

(GnRH)

polyphosphoinositides.

acting

cultured

hormone

of

turnover

activation

Life

8-bromo

releasing

IPl,

Ca2+ -mobilizing

phosphoinositide second

inositol

inositol

by further kinase

the

and

cGMP formation.

protein

formation

stimulatory to

of

analog prelabeled

enhanced

additive

of

permeable

Hlinositol

The

inhibition

(IP2)

as a modulator

in (IPl).

hydrolysis

mobilizing

and

of gonadotropin

1,4-bisphosphate

and

to

analog

IPl

stimulates

the

stable

monophosphate

accumulates

for

the

GMP (8-BR-cGMP)

cyclic

George S. Wise Faculty of Ramat Aviv 69978, Israel

1990

Addition

inositol

also

Biochemistry, The Tel Aviv University, 5,

pituitary

Naor

biological bidirectional

protein

kinase

systems

(e.g. control

Vol.

167,

No.

3, 1990

mechanisms coupled

BIOCHEMICAL

were to

described,

cyclic

no

inositol

GnRH, retie

(5-8).

Ca2+

in

the

pituitary

stimulated

phosphoinositide

sponsible

for

logical

turnover

postulate

that

and

MATERIALS

AND

medium cin

cells

199,

(100

ate

KRBG)

and

Carlos,

CA) or points

rapid

pituitary

ligand-

cells

Ca2+.

is

re-

Since

(14),

the

any physio-

To

to

phosphoi-

that

end,

effect

days

were

old)

we

on ligand-

cells/dish)

then

were

penicillin

for

8-Bromo-cGMP other

as indicated.

test

twice

with

(Sigma), substances The

cultured

for

assay 983

3 days

14

(2 mg/ml) without

GnRH were

Li+

was terminated

Ci/mmol,

bicarbon-

and

(Peninsula then

in

streptomy-

in Krebs-Ringer

or

Wis-

described

(100 units/ml),

glucose

15 min

from

(5 uCi/ml,

washed

containing

prepared

as previously

myo-[2-3H]inositol

were

incubated

KRBG.

very

be secondary

cells

(28

serum,

and

pH 7.4

37OC in

time

rats

5% horse

Cells

buffer,

well

a modulatory

pituitary

(5~10~

pg/ml,

Amersham).

GnRH and TRH is

METHODS

female

(15) * The

for

the

hand

role

(12,13).

of

exert

where

turnover.

anterior

tar-derived

systems

Ca2+-dependent

mobilization

pitui-

a role

mobilization

cGMP will

cultured

other of

in

to

phosphoinositide

On the

turnover

cGMP might

Cultured

in

natriu-

no physiological

actions

cGMP is

stimulated

there signal-

biological

that

attributed

nositide

(8,9).

Ca2+

pituitary

role

most

was suggested

cellular

of

as in

known

It

of

To date

and atria1

cGMP formation

cGMP is

(10-12).

formation

(TRH)

was demonstrated,

documented

inhibition

potentiating

hormone

stimulate However,

mediating

receptors

formation.

cGMP formation

pituitary

in

(2-4).

nucleotides

releasing

(ANF))

cells

enhanced for

thyrotropin

COMMUNICATIONS

of

resulted

turnover

cyclic

phosphate

factor

tary

of

RESEARCH

activation

formation

phosphoinositide

examples

induced

BIOPHYSICAL

whereupon

nucleotide

ligand-stimulated are

AND

BSA (O.l%, (10 mM)) Lab.,

introduced by the

at San for

addi-

Vol.

167,

No.

3, 1990

BIOCHEMICAL

tion

of

1 ml cold

were

harvested

ice,

and 2 ml of

vortexed

to

by scraping

chloroform/HCl

5 ml of

Ins-P

(formate

were resolved

form)

phase

added

each fraction

was determined

glass

added.

partition. the

and the

samples

tubes

in

analysis

upper

of

phase

were

was

neutral-

on Dowex 1x10 columns

(15).

by liquid

Cells

Samples were

For

(Ins-P),

described

(1 ml).

to

were

by chromatography

as previously

COMMUNICATIONS

medium

transferred

phosphates

H20 were

RESEARCH

incubation

(1OO:l)

for

inositol

collected,

the

BIOPHYSICAL

and were

and centrifuged

water-soluble

ized.

methanol

AND

The

[3H]-content

scintillation

of

counting.

RESULTS After

prelabeling

3H]inositol or for

for

3 days,

comparison

Table 1: --

cultured

with

cells

were

GnRH in the

cells

stimulated

presence

with with

myo-[28-Br-CGMP,

or absence

of

Li+

Effect of 8-Br-d;Mp and &RB on [3B]In.s-P production in the presence or absenceof Li+ (cpn/dish)

Addition.s

Li+

IP1

ccoltrol 8-Br-OGMP

-

GmH Control

+

a-Br-d;Mp

+

QlRH

+ Cultured

prelabeled

with

treated

with

without

8-Br-cGMP

min.

the

pituitary

Ins-P

IP2

IP3

361*14

75* 7

60+10

3aa+i9

67* 7

78*22

615*49**

go* 5*

89*

617*68

128212

100*25

1010*63**

133*29

116*17

1651*228**

211*16*X

190*15**

pituitary

cells

myo-[2-3H]inositol

or without

(5~110~ for

Li+ (10 mM) for

were extracted shown

triplicate.

* p x0.05;

are

and separated

mean + SE of **

p < 0.01. 984

three

cells/

3 days.

dish)

Cells

were

were then

15 min and later

(1 mM) or GnRH (100 nM) for

The results

4*

with

an additional

or 20

on Dowex 1x10 columns. experiments

each

done

in

(10

Vol.

167,

No.

3, 1990

BIOCHEMICAL

mM) which

is

known

to

block

As shown

in

Table

1,

and

here,

8-Br-cGMP

IP2,

effect

incubation

is

presence

or

absence

8-Br-CAMP

production

of

(results

shown

[ 3H]IPl

study in

Li+

Fig.

in

the

formation

formation 1).

for

cGMP-stimulation 8-Br-cGMP,

presence

of

in

IPl,

not

Li+

the

hypophysiotropic

IP2

and IP3

in

similar

condi-

under

effect

The

of

10 (p < 0.05)

not

stimu-

1).

but

but

and the

Also,

and Fig.

shown

inositol,

and consistent

shown

not

(p