Serum, bradykinin and vasopressin stimulate release of inositol phosphates from human fibroblasts

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 123, No. 2, 1984 September

Pages

17, 1984

Serum,

Bradykinin And Vasopressin Stimulate Release Inositol Phosphates From Human Fibroblasts Lucia

M. Vicentini

and Mitchel

Department of Pharmacological The University of Chicago, Received

July

26,

663-670

Of

L. Villereal

and Physiological Chicago, Illinois

Sciences, 60637

1984

The mitogens serum, vasopressin and bradykinin stimulate a significant rise in the inositol phosphate content of cultured human fibroblasts within 10 seconds, while serum- and bradykininstimulated arachidonic acid release does not occur until after 30 seconds. The release of inositol phosphates is not secondary to a rise in Ca activity since the Ca ionophore ionomycin does not stimulate release of inositol phosphates. Moreover, we show that phospholipase C in human fibroblasts is activated by these mitogens at resting Ca levels since TMB-8, which blocks the mitogeninduced rise in Ca activity, does not affect the serum-stimulated accumulation of inositol phosphates.

Mitogenic series

of

cell

membrane

division.

exchange our

laboratory human

identify

to the

the

activation

shown that in

the

phospholipase

Abbreviations: benzoate;

has

studied

the

of

of

is mediated

Ca+2 mobilization of

these

appears

the

TMB-8, BSA, bovine

lead

Na+/H+

of

exchanger.

Na+/H+

exchange

(3).

to be involved

in

exchange

in

the

order

binding

is a necessary

step

(1‘2)

this

and that

phospho-

regulation

HSWP cells.

mitogen

in

we have

In addition, the

in

mitogen

Previously

exchanger

by calmodulin

block

events,

Na+/H+

couple

Ca+2 mobilization the

to Na+/H+

(HSWP) extensively which

a

eventually

mitogen-stimulated

regulation

events

and Na+/H+ activity

early

fibroblasts

biochemical

that

initiates

of an amiloride-sensitive

one of

foreskin

fibroblasts

events

is

stimulation

activity

cultured

activation

an intracellular

Ca+2 effect lipase

of

and cellular

Since

system

cultured to

stimulation

of both Inhibitors

stimulation

of

Na+

8-(N,N-diethylamino)octyl-3,4,5-trimethoxyserum albumin. 0006491X/84 663

All

Copyright 0 1984 rights of reproduction

$1.50

by Academic Press, Inc. in any form reserved.

Vol. 123, No. 2, 1984

influx

BIOCHEMICAL

in HSWP cells

(4) and melittin, a rise

stimulates

lipaSeS,

Cat2

activity

(6).

pase

activity

in our

lowing no

the

mitogens.

in

However,

release

information

that

mitogenic

late

arachidonic

Na+ influx

previous of

an activator

since

the

several

(4,5)

all

Cat2

studies

factors

such

as serum,

acid

release

appear

seemed

to mobilize

important

viously

via

and/or

PDGF and bradykinin

stimu-

mobilization

C. Thus

like to

the

assess

HSWP cells recently

and for

in

phophoinositides

nature to method

(inositol are

a rise

HSWP cells

the

mitogenic for

direct

phosphates)

generating

and serum (lo),

activity

it pre-

is phospholi-

measurement released

for

cellular

protein

stimulation,

by

a signal

important

phospholipase

hydrolyzed

. Baterlals

intracel-

of phosphatidylinositol

of other

of

of

has

in HSWP cells

phospholipid-dependent

Cat2,

response

available products

the

activation

phospholipase

phospholipids

phospholipase

the hydrolysis thereby

of

bradykinin

Cat2

polyphosphoinositides,

components

soluble

induce

by

proposed

in mitogen-stimulated

would

we had

has been

an elevation

the

by fol-

acid,

it

and vasopressin,

whether

phospholi-

was activated

(7,8)

intracellular

to test

implicated

pase C, which

Cat2

(9),

of

phospholipase

recent

activity

and intracellular

t3Hlarachidonic

which

phospho-

(4) was monitored

of inositol-containing A29 Since the breakdown been demonstrated for agonists which produce lular

of

stimulation

studies

incorporated

concerning In

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

kinase

activated

in

we applied

the

of

the

water

when

the

phos-

phospholipase

C.

liuuinethods

Human fibroblasts (HSWP), derived from foreskin, were obtained from James Regan, Oak Ridge National Laboratory. They were cultured in Eagle's minimal essential medium (EMEM; Kansas City Biological, Inc., Lenexa, KS) containing 10% fetal bovine serum (FBS; Kansas City Biological Inc., Lenexa, KS). Cells were grown at 37 OC in a 95% air, 5% co2 atmosphere and were used between the 10th and the 25th passages. Cells were removed from stock flasks by trypsinization and were seeded onto 60 mm culture dishes for use in the experiments. The cells were used for the experiments at 3-5 days after subculturing while they were in the 664

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 123, No. 2, 1984

logarithmic phase of growth. Twenty four or 36 hrs before the the cells were labelled with 1 uCi/ml of [3ff]myoexperiment, inositol (NEN 15.8 Ci/mmol) in inositol-free BME (basal medium The day Eagle modified for diploid cells) containing 0.1% FBS. of the experiment the cells were rinsed three times in Hepes inositol-free Hank's salts medium and incubated for buffered, different time periods in the presence or absence of stimulating The method for the determination of agents and of 10 mM LiCl. the total inositol phosphates is the one described by Berridge et al. (11). Briefly, after the various treatments, the medium was aspirated and the cells were extracted with chloroform/methanol (1:2). Water was added to separate the phases and a portion (1.3 mls) of the upper aqueous phase was removed and diluted to 3 mls with water. For the analysis of the total inositol phosphates, a 50% (w/v) slurry of AG resin (1x8 100-200 mesh, Biorad. Laboratories, Richmond, CA) in the formate form was added to the aqueous phase, the resin was washed 3 times with 5 mM myo-inositol and the inositol phosphates were eluted with 0.6 ml of 0.1 M formic acid /l.O M ammonium formate. One half ml of this eluate was counted after adding 5 mls of scintillation cocktail. [3H]Arachidonic acid release was assayed after incubating the cells in Hepes-buffered, amino acid free EMEM containing 0.1% FBS for four hours. During the final hour of serum deprivation, the incubation medium contained 1 ciCi/ml of 13H]arachidonic acid (NEN). The cells were then rinsed five times with 3 mls of Hepes buffered medium and incubated for various times in Hepes buffered EMEM in the presence or absence of stimulating agents. After the treatment, an aliquot of the medium was taken from the dishes, centrifuged and the supernatant counted in vials containing scintillation fluid. The cells were extracted with chloroform/methanol (1:2) and then water, acidified with formic acid, was added. Additional chloroform was added and the samples were centrifuged to separate the phases. After centrifugation, the organic phase was collected and dried under nitrogen. It was then redissolved in chloroform and applied to silica gel coated glass plates (Type arachidonic acid was spotted in an adjacent lane '3 . Unlabeled to serve as a standard. The solvent system was ethylacetate/ trimethylpentane/acetic acid/water (11:5:2:10]. After the plates were developed, the spots were visualized with iodine vapor, the arachidonic acid spots were scraped and the silica was counted after adding to it 400 111 of water and 4 ml of scintillation fluid.

Fig.

1 shows

100 nM bradykinin ~3H]inositol

serum,

increase

in

time

for

in 24-36

bradykinin the

levels

or of

at 30 seconds

is 2.8

for

and 1.8 times

bradykinin

course

of

and 100 nM vasopressin

phosphates

been prelabelled with

the

times

HSWP cells. hrs with

the

on the

the

[3Hlinositol

for

there

level

vasopressin. 665

content

were

phosphates.

basal

of

When HSWP cells

vasopressin, inositol

effect

was a

10% serum, of total that

had

stimulated dramatic

The stimulation for

serum,

2.6

times

At 1 and 5 minute

BIOCHEMICAL

Vol. 123, No. 2, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Effect of serum, bradykinin and vasopressin on the 1. ASWP levels of total [3H]inositol phosphates in HSWP cells. cells were labelled with [3R]inositol as described in the Material and Methods section. Cells were incubated in 10% serum (O), 100 nM bradykinin (A) or 100 nM vasopressin (m) for various times. The cells were extracted and analyzed as described in the Material and Methods section. Results are expressed as means + S.E.M. of % increase over basal levels from four different experiments.

EisL

serum

stimulations,

vasopressin

in

10 mM LiCl in order

to

of

which

the

to the has often

stimulation,

Although phates inositol significant

which not into

of

presumed these to

shown here,

in

during

the

Thus,

to be the mitogens

the

we have

and have inositol

is

clear

shown

that

in

release,

of phopholipase

A2

phospholipase

C

the

inositol

the

that

acid

inositol

fractionated

bisphosphate 666

of

time

and magnify

stimulates

release

than

incubation

it

result

potent

phosphates.

on arachidonic

monophosphate,

trisphosphate rise

inositol

phosphatase

known effect

leads

inositol

of the

phosphates.

already been

medium

myo-inositol

inositol

each

activity

in the

the

block

seem to be more

accumulation

inducing

was present

increase addition

and bradykinin

phosphates. inositol

phos-

bisphosphate mitogens

and inositol

produce trisphos-

and a

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 123, No. 2, 1984

Table I. Content of [3BlinOSitOl phosphates in HSWP cells: of ionomycin on basal cells and TMB-8 on serum-stimulated Inositol phosphates (% of control)

Assay condition Serum-free

Effect cells

control

Serum control Serum + TMB-8

100 +

27

880 +

89*

1000 * 110*

Ionomycin

0.1

Ionomycin

0.5 /.Gl

Ionomycin

1.0

pM

122 +

22

135 f.

20

93 +

32

PM

HSWP cells were labelled with [3H]incsitol as described in the Material and Methods section. Cells were stimulated for 5' with 10% serum with andwithout 50 LIM TMB-8 or in the secondseriesof experiments in the absence of serum with increasing doses of ionomycin. The cells were extracted and analyzed as described in the Material and Methods section. Results are expressed as % of basal levels + S.E.M. of triplicate determinations for a representative experiment. * Significant to the p < 0.01 level.

phate

levels

within

5 seconds

(Jamieson

and Villereal,

in

prepa-

ration). To determine on the with

release the

of inositol

Ca+2 ionophore

ionomycin,

stimulation presence

of

phates.

a rise

mitogens our

intracellular

50

release

From

these

PM TMB-8,

last

Ca+2

can activate

knowledge,

this

activity

(6),

shown

and

first

that

of

HSWP cells 1 show that

human

C at resting demonstration

any

Moreover to block

appears is

to

induce

not

the on the

[3H]inositol it

in

mitogens

had no effect

phosphoinositides

phospholipase is the

Table

does not

has been

experiments,

of

the

phosphates.

accumulation

two

of

Ca+2 activity

[3Hlinositol which

in

(6),

in HSWP cells

breakdown in

HSWP cells of

serum-stimulated

mitogen-induced to

increase in

effect

we treated The data

Ca+2 rise

mitogen-induced 5 minute

the

the

ionomycin.

levels of

of

phosphates,

at doses which

mitogen-stimulated

the

Ca+2-dependence

the

phosthat

the

secondary

fibroblasts Ca+2 levels. of a phospholipase

the TO

BIOCHEMICAL

Vol. 123, No. 2, 1984

C stimulation

in

the

total

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

absence

of

a rise

in

intracellular

Ca+2. In

previous

stimulate in

studies

release

show that

was the

the

the

release shown).

is

medium

is

bradykinin

Table

of

release

over

changed only

at after

stimulation

30 seconds of the

cells

A2 activity

at in

An increase

in

control

for not

in

phosphates

( data

not

present

in

serum

and Con-

shown).

Assay condition

(cpm/dish)

Arachidonic medium (cpm/ml)

serum-free

554 + 35

57 + 30

344 + 30

bradykinin

736 + 56*

57 + 15

390 * 20

192 + 60

331 + 50

180 + 40

330 +

BSA

serum

866 + 72*

acid cell (cpm/dish)

8

HSWPcells were labelled with [3H]inositol or [3H]arachidonic Cells acid as described in the Material and Methods section. were incubated for 10 seconds with 10% serum or 100 nM bradykinin. After the incubation, an liquot of the medium was counted for the determination of the 1BHlarachidonic acid release. The r5sidual medium was aspirated and [3H]inositol phosphates and the [ Hlarachidonic acid were extracted and analyzed as described in the Material and Methods section. Results are means + S.E.M. of triplicate determinations. This is a representative experiment out of three. * Significant to the p < 0.05 level.

668

the

arachidonic

acid

levels

while

II: Effect of serum and bradykinin on inositol phosphate levels and (3R]arachidonic release in HSWP cells Inositol

we

serum and

and

stimulation

(data

2,

10 seconds, cells

[3H]arachidonic

respectively

for

the

of

Table

phosphates

1.6 times

both

time.

arachidonic

inositol

levels

190% and 150% over

stimulated

the

In

the

already

release

However,

phospholipase

of

basal

that

fibroblasts.

whether of

and bradykinin

C activity.

is

acid

seen

At 5',

result

phospholipase

bradykinin

not is

direct

serum

from

ascertain

and bradykinin

[3H]arachidonic

medium

the

for

to

release

not

stimulation

serum

1.3 times

acid

we did

or was secondary

by both

(4) we showed that

[3H]arachidonic

those

acid

studies

Vol. 123, No. 2, 1984

trols

for

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

serum

showed

that

through

a mechanism

pase

lation

This

of

sitides

of

acid

phatidylinositol

cycle

Of a specific these

activation

is

very

early

a stimulation

of

pancreatic

observation

that

this

data

are

that

inositol

may be a key event particularly the

actions

of

in

the

interesting phosphorylation two

suggests

an important

regulating

cellular

and

the

is

fibroblast process

in

view

oncogene for

proliferation.

the

that

the

phospholi-

its

one of the is

subsequent results

mobilization

of

recent

Ca+2

results

trisphosphate (12,13,14),

the

stimulated

by 3

system

suggests

of mitogenesis. of

the

of phosphatidylinositol

separate role

the

release the

would

previous

mobilization

Ca+2 in

action

of fibroblasts

inositol

phosphate

phos-

~2 but

that

our

implicating Ca+2

the

by the

and the

by mitogens,

cells

known to mobilize

implicating the

of Na influx

in

to

appears

given in

Arach-

explanation

C activity Thus,

of

acid

stimulation

activity

messenger

it

be broken

acid.

component

comparison

case

phosphates.

and liver

as a second

in

mitogen

phospholipase

and activation

mitogens

delayed

phosphoino-

then

phospholipase

phospholipase

of inositol

implicating

do activate

following

could

by the by a stimu-

on the

An alternative

In either

events

stimulated

phosphatidic

the

and bradykinin

arachidonic

another

from A2.

temporally

C stimulation.

in

i.e.

agents

pase

release

from

phospholipase

be that

acting

to release

derive

HSWP cells

be explained

which

lipase

in

acid

by BSA

on phospholi-

by serum

could

studies

increased

that

stimulated

diacylglycerol,

can also

is

suggest

pattern

previous

of effects

C activity

down by diacylglycerol idonic

data

release

produce

release

[3Hlarachidonic

phospholipase

to

BSA since

independent

phosphates

release

same agents.

is

These

inositol the

acid

which

(4).

of

precedes

contained

[3Hlarachidonic

activity

release

response

products

phosphatidylinositol

Our

recent

findings as one of

(15,161

which cycle

in

Bl(?ZHEMlCAL

Vol. 123, No. 2, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

GEMENTS We thank Kathy Toscas for her excellent technical assistance Della Akres for her help in preparing the manuscript. This work was supported 01182 (MLV). Preliminary reports in Fed. Proc. u,496.

1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

by

of this

NIH

grant

study

GM 28359

were published

(MLV)

and

and RCDAAM

in abstract

form

Villereal, M.L. (1981) J. Cell. Physiol. lpz, 359-369. Owen, N.E. and Villereal, M.L. (1982) Biochem. Biophys. Res. Commun. Lpp, 762-768. Owen, N.E. and Villereal, M.L. (1982) Proc. Natl. Acad. Sci. U.S.A. n, 3537-3541. Vicentini, L.M., Miller, RJ. and Villereal, M.L. (1984) J. Biol. Chem. m, 6912-6919. Rozengurt, E., Gelehrter, T-D,, Legg, A. and Pettican, P. (1981) Cell & 781-788. Mix, L.L., Dinerstein, R.J. and Villereal, M.L. (1984) Biochem. Biophys. Res. Commun.m, 69-75. Hong, S.L. and Deykin, D. (1981) J. Biol. Chem. X&., 52155219. Shier, W.T. and Durkin, J.P. (1982) J. Cell. Physiol. ll2, 171-181. Mitchel, R.H. (1975) Biochim. Biophys. Acta. m, 81-147. Owen, N.E. and Villereal, M.L. (1983) J. Cell. Physiol. X2, 23-29. Berridge, M.J., Dawson, R.M., Downes, C.P., Heslop, T.P. and Irvine, R.F. (1983) Biochem. J. 212, 473-482. Streb, H., Irvine, R.F., Berridge, M.J. and Schulz, I. (1983) Nature m, 67-69. Burgess, G.M., Godfrey, P.P., McKinney, J.S., Berridge, Irvine, R.F. and Putney, Jr., J.W. (1984) Nature g,'63-66. Joseph, S.K., Thomas, A.P., Williams, R.J., Irvine, R.F. and Williamson, J.R. (1984) J. Biol. Chem.m, 3077-3881. Sugimoto, Y., Whitman, M., Cantley, L.C. and Erikson, R.L. (1984) Proc. Natl. Acad. Sci. U.S.A. BL, 2117-2121. Macara, I.G., Marinetti, G.V. and Balduzzi, P.C. (1984) Proc. Natl. Acad. Sci. U.S.A. &l, 2728-2732.

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