Endothelin stimulates phospholipase C in cultured vascular smooth muscle cells

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 30, 1988

Pages 1360-1368

ENDOTHELIN STIMULATES P H O S P H O L I P A S E C IN CULTURED V A S C U L A R SMOOTH MUSCLE CELLS Th~r~se J. Resink, Timothy S c o t t - B u r d e n and Fritz R. B~hler of

Department

Research, Hypertension Laboratory, Hospital, 4031 Basel / Switzerland

University

Received November Ii, 1988

SUMMARY. Cultured vascular smooth muscle cells from bovine and rat thoracic aortae and from human omental vessels have been examined for cellular responses to endothelin. In m y o - [ 3 H ] - i n o s i t o l - p r e l a b e l led cells endothelin induced a rapid (within 30 sec) and p r o t r a c t e d increase of [3H]-inositol content in inositol bis- and tris-phosphates. Concomitantly, significant polyphosphoinositide hydrolysis occurred within 30 sec. A c c u m u l a t i o n of [3H]-inositol m o n o p h o s p h a t e and hydrolysis of p h o s p h a t i d y l i n o s i t o l were delayed. In cells p r e l a b e l l e d with [3H]-arachidonic acid endothelin promoted rapid production of [3H]-diacylglycerol which decayed slowly toward control values after reaching m a x i m u m levels (1-2 min). Halfmaximally effective concentrations of endothelin for all these cellular responses were comparable (N 3-7 nM) and not significantly different between the vascular cell isolates. The involvement of the phospholipase C-signal transduction pathway in m e d i a t i n g endothelininduced v a s o c o n s t r i c t i o n is invoked. ©1988AcademicPress, Inc.

INTRODUCTION.

Endothelin

endothelial cells tion in

a

21-amino

acid

(i), induces a potent

a variety

of isolated

peptide

derived

from

and prolonged vasoconstric-

blood vessels from both man

(2) and

experimental animals

(1,3,4). Functionally diverse compounds such as

calcium antagonists,

protein kinase C inhibitors and those elevating

intracellular cyclic nucleotides promoted

are

able

vasoconstriction

(1,2,5).

While

influence of endothelin on

numerous

signal

Abbzeviations. Ins-P3, Ins, phate,

reverse endothelin-

such

and Ptd-InsPz,

and tris-phosphate,

respectively.

1360

suggest

an

Ins-P, Ins-P= and respectively;

phosphatidyl-inositol,

and -inositolbisphosphate,

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

data

transduction pathways,

VSMC, vascular smooth muscle cells;

inositol mono-, bis-,

Ptd-InsP

to

Ptd-

-inositolphos-

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

there is presently a paucity of information concerning the nature of cellular biochemical responses to endothelin. Characteristic of some v a s o c o n s t r i c t i v e

agonists is their ability

to elicit phospholipase C mediated d e g r a d a t i o n of p h o s p h a t i d y l i n o s i tolbisphosphate process,

(6,7), and at least two

namely inositol triphosphate

Ca 2÷) and diacylglycerol

(which

involved

a

in

mediating

essential for the control vascular smooth

catabolic products

(which mobilizes intracellular

activates

complicated of

protein

cascade

contraction

muscle cells

of this

shown to

kinase

C), are

of cellular events

(8,9).

In

cultured rat

possess specific e n d o t h e l i n

receptors, endothelin promoted an elevation of intracellular Ca 2+ but apparently had no influence on inositol lipid m e t a b o l i s m (i0). The Ca2+-response was only EGTA or calcium antagonist lar Ca 2÷ mobilization. tion by

p a r t i a l l y inhibited

in the

presence of

(10), thus indicative of some intracellu-

This,

together with reversal of v a s o c o n s t r i c -

the protein kinase C inhibitor H-7

involvement of phosphoinositol

(5) strongly invokes the

lipid m e t a b o l i s m in

cellular respon-

ses to endothelin. In

this

study

we

present evidence that endothelin does indeed

stimulate p h o s p h o i n o s i t i d e hydrolysis muscle cells from human,

in

cultured

vascular smooth

rat and bovine sources.

MATERIALS AND METHODS Materials. With the exception of fetal calf serum (Fakola AG, Switzerland) all tissue culture material and chemicals were from Gibco AG, Switzerland. The radioisotopes myo[2-3H]inositol (16 Ci/mmol) and [ 5 , 6 , 8 , 9 , 1 1 , 1 2 , 1 4 , 1 5 - 3 H ] a r a c h i d o n i c acid (195 Ci/mmol) were purchased from Amersham, FRG. Porcine e n d o t h e l i n was from Peptide Institute Inc., Japan. Dowex I-X4 was from Bio-Rad, Switzerland and Silica Gel 60 plates from Merck, FRG. All other chemicals were of the highest commercial purity. Cell culture: Vascular smooth muscle cells (VSMC) from rat thoracic aorta, bovine thoracic aorta and human omental vessels were isolated, cultured and c h a r a c t e r i z e d as d e s c r i b e d p r e v i o u s l y (11,12,13). VSMC were used at confluence between passage 4 and i0 ° . Phosphoinositol lipid metabolism: Confluent VSMC were p r e l a b e l l e d for 48 hrs with m y o - [ ~ H ] - i n o s i t o l (5 ~Ci/ml) under serum- and inositol-free conditions. VSMC were then washed (3x3 ml) and preincubated for 30 min at 37°C in 1.0 ml isotonic phosphate b u f f e r e d saline containing 20 mM TES/HEPES (pH 7.3) and 30 mM LiCl. VSMC were exposed to endothelin at concentrations and for periods indicated in figures and table. Incubations were terminated by aspiration of buffer and addition of 1.0 ml CHCI3:MeOH:HCI (1:2:0.05; v/v). Extraction and chromatographic (Dowex I-X4) separation of inositol phosphates and phosphoinositol lipids (after deacylation) was p e r f o r m e d as p r e v i o u s l y described (7,14). Production of diacylglycerol: Confluent VSMC were rendered q u i e s c e n t by culture for 48 hrs in serum-free medium (containing 0.1% w/v 1361

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

bovine serum albumin in place of fetal calf serum). VSMC were then p r e l a b e l l e d with [3H]-arachidonic acid ( 1 ~Ci/ml) for 3 hrs in the presence of minimal essential m e d i u m containing 20 mM TES/HEPES (pH 7.3) and 0.1% (w/v) bovine serum albumin. VSMC were washed (3x3 ml) and incubated at 37°C for i0 min in 1.0 ml of the same m e d i u m prior to exposure to endothelin at c o n c e n t r a t i o n s and for periods indicated in figures. Reactions were terminated by aspiration of medium and addition of 1.0 ml CHCI3:MeOH (1:2; v/v) and lipids extracted as described (7). D i a c y l g l y c e r o l was resolved on h e a t - a c t i v a t e d Silica plates using the solvent b e n z e n e : d i e t h y l e t h e r : a m m o n i a (100:80:0.2; v/v) as described (7), visualized by iodine staining and identified by comparison with simultaneously chromatographed standard. The appropriate area was scraped off into vials containing 1.0 ml toluene, and samples shaken gently for 1 hr prior to d e t e r m i n a t i o n of radioactivity. All experiments were performed using triplicate wells for each determination, and where appropriate, Student's ttest for unpaired data was applied to determine statistical significance.

RESULTS In quiescent

and m y o - [ 3 H ] - i n o s i t o l

prelabelled VSMC from human,

bovine and rat vessels endothelin induced a rapid least <

(Fig. IA). This

effect of

(Fig. IB) and half-maximal calculated

(mean±SD)

to

be

for

bovine,

[3H]-inositol

content

phosphates

indicated

in

(p<0.001)

Increases of (p<0.05)

after

accumulation three

to

i0

[~H]-inositol

6.0±3.1 nM

respectively.

Analysis of

VSMC min

of

300-350% above control

and

exposure

content

inositol

min)

(Table i). Highly

in

such

Ins-P

levels of

(zero time,

responses were

to endothelin were

(Table i).

only marginal

whereas after 10 min, [3H] in

100%)

this fraction

(Table I). The rapid

[3H]-Ins-P2 and Ins-P3 was

loss of [3H]-inositol (i0

resolved

Ins-P2 and Ins-P3 occurred

types

30 sec exposure to endothelin,

(30 sec) accumulation of

the sustained

of the peptide and

inositol phosphates

and in the presence of 30 mM LiCl,

a concomitant

nM

chromatographically

significant

up

4.0±1.2

endothelin promoted increases in mono-(Ins-P),

within 30 sec for all for

nM,

rat and human VSMC,

and tris-(Ins-P3)

protracted

endothelin was d o s e - d e p e n d e n t

stimulatory concentrations 2.5±1.0

bis-(Ins-P~)

were

(p at

0.01 after 30 sec) accumulation of [3H]-content in inositol

phosphates

were

and marked

associated with

from p o l y p h o s p h o i n o s i t i d e s while

accumulative

response

to

endothelin was

associated with a decrease in [3H]-inositol content of only phosphatidylinositol

(Fig. 2).

Exposure of quiescent endothelin resulted dent production of VSMC.

Significant

[~H]-arachidonic

in a time-

(Fig.

[3H]-diacylglycerol (p at

least < 1362

acid p r e l a b e l l e d

3A) and dose-

0.01)

in

bovine,

VSMC to

(Fig. 3B) depenrat

and human

increases in diacylglycerol

V o l . 157, N o . 3, 1 9 8 8

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

600

500

.c 400 "5

B

~o300

`5 300

o

c

o 200

~" 200

c

100

.................... i

i

0

2

i

1

4 6 Time (rain)

100

i

i

!

B

10

0

/tf

i

i

I

i

i

10-1° 10-9 10"~ 10-7 Endothetin (M)

10-6

Figure i: Time- and dose-dependent accumulation of inositol phosphates in VSMC exposed to endothelin. Myo-[3H]-inositol prelabelled VSMC from human ( O ), rat ( • ) and bovine { ~ ) vessels were exposed to 10 -7 M endothelin for the indicated times (Panel A) or to various concentrations of endothelin for 1 min (Panel B). Data (mean±SD, where at least 3 separate experiments were performed in every case) represent the percentage increase in [~H]-content in inositol phosphates (comprising mono-, di- and tris-inositol phosphates) relative to that present (100%) in VSMC at time zero (Panel A) or without exposure to endothelin (Panel B). [3H]-inositol content in the three VSMC species remained constant (±5%) when incubated for given times without exposure to endothelin (Panel A, ----). Halfmaximally effective endothelin concentrations (given in text) were determined for each individual dose-profile using computerized weighted non-linear regression analysis (15).

occurred min,

within

30 sec a n d m a x i m u m

thereafter

maximally exposure 6.4±2.2

decaying

effective to

various

toward

levels control

concentrations, doses

nM a n d 7 . 5 ± 2 . 9

were

values

estimated

of e n d o t h e l i n

n M for b o v i n e ,

obtained

(Fig.

within

(Fig.

3A).

following 3B) w e r e

rat a n d h u m a n

Half1

min

7.2±3.0

VSMC,

1-2

nM,

respecti-

vely.

DISCUSSION The present that

study using

endothelin

human,

potently

hydrolysis

of

inositol

evidenced

by

production

of b o t h

bovine

stimulates

phospholipids.

hydrolysis inositol

of

a n d rat V S M C phospholipase

Such

a

response

phosphoinositides

phosphates 1363

demonstrates

and

C-mediated was

clearly

concomitant

and diacylglycerol.

The

early

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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V o l . 157, N o . 3, 1 9 8 8

,oo[

B I O C H E M I C A L A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

Ptd- [nsP

Ptd-[nsP 2

Ptd-Ins

u

o 60

~

~,

30 sec.

10 min

30 sec

10 rain.

30sec.

10 rnin

Figure 2: Endothelin-induced phosphoinositide hydrolysis in VSMC. [~H]-inositol content in phosphatidylinositolbisphosphate (PtdInsP2), phosphatidylinositolmonophosphate (Ptd-InsP) and phosphatidylinostol (Ptd-Ins) was determined after incubation of VSMC without or with 10 -7 M endothelin for 30 sec or 10 min. Experimental procedures are given in Materials and Methods. Data (mean±SD) express the percentage of [~H]content in deacylated inositol phospholipids where that present in control samples was taken as 100% for each different phosphoinositides. ([3H]content fn each did not vary significantly (±5%) for VSMC incubated without endothelin). The numbers of separate experiments performed for h u m a n ( • 5, rat ( [] ), and bovine ( [] ) VSMC were 4, 3 and 3, respectively. The asterisks indicate where the p value of significance between VSMC incubated without or with endothelin was at least < 0.01.

700

700 B

o

'.-E .c "5

8> , 350 cJ

~5 i

"-r

~

100

.

.

.

.

.

.

.

.

100

.

;

i

1'o

0

Time (mini

ff

i

i

i

i

10"1° 10-9 10-8 10-7 Endothelin (N)

i

10-8

Figure 3: Endothelin promotes diacylglycerol production in VSMC. [3H]-arachidonic acid - prelabelled VSMC from human ( O ), rat ( • ), and bovine ( ~ ) vessels were incubated with 10 -7 M endothelin for various times (Panel A5 or with various concentrations of endothelin for 1 min (Panel B). Data (mean±SD, where 3 separate experiments were performed in every case) represent the % increase in [~H]diacylglycerol relative to that present (100%5 at time zero (Panel A) or without exposure to endothelin (Panel B). [~H]-diacylglycerol levels remained steady (±5%) in all VSMC isolates w h e n incubated for the indicated times in the absence of endothelin (Panel A, .... ). Dose-profiles were analyzed individually (15) to determine halfmaximal stimulatory concentrations of endothelin (given in text). Absolute values (dpm per 106 cells) for [3H]-diacylglycerol in control VSMC (zero time in Panel A, and in absence of endothelin for Panel B) were 175±38 (n=65, 197~24 (n=65, and 256±29 (n=65 for human, rat and bovine isolates, respectively. 1365

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

kinetic characteristics response in

of

the

endothelin-induced phosphoinositide

VSMC are entirely compatible with those established for

other p h o s p h o i n o s i t i d e - m o b i l i z i n g types

hormones

in

a

variety

of oell

(6-8).

Our observations contradict those of Hirata et al (i0) who failed to observe accumulation of VSMC exposed

(total)

to endothelin

(and in the presence of 10 mM LiCI)

20 min,

and thus concluded that

play a

major role

p h o s p h o i n o s i t i d e breakdown

for

may not

in e n d o t h e l i n - i n d u c e d elevation of intracellular

Ca z+ ([Ca~+]i). We are Hirata et

inositol phosphates in rat aortic

al (10)

unable to

explain the

negative findings of

in relation to inositol phosphates

able to

reproduce our

However,

these

findings using

and have been

their incubation conditions.

same authors demonstrated both transient and sustai-

ned [CaZ+]i responses of VSMC to e n d o t h e l i n of which only the latter could be nist

inhibited in

the presence of EGTA or Ca2+-channel antago-

(10). We propose that the present d e m o n s t r a t i o n

induced

production

of

inositol

triphosphate,

of endothelin-

which recruits Ca 2+

from intracellular Ca2+-stores

(8),

the transient

by endothelin that occurs even in the

[Ca=+]i signal

might account

absence of extracellular Ca =+ or CaZ+-influx

for induction of

(i0).

Our results would suggest that diacylglycerol most likely derives from

the

polyphosphoinositides

since

its

generation

coincides with hydrolysis of Ptd-InsP2 rather than that The delayed

temporally of Ptd-Ins.

stimulated loss of Ptd-Ins may reflect its phosphoryla-

tion by kinase{s)

to replenish the

by p h o s p h o l i p a s e

C (8,16),

VSMC a late onset

of direct

polyphosphoinositides

although,

in

hydrolysed

angiotensin II stimulated

Ptd-Ins hydrolysis

by phospholipase

C

has been proposed to account for sustained diacylglycerol p r o d u c t i o n (7). The latter might also be true for endothelin since diacylglycerol

formation

in

VSMC

transient diacylglycerol (17))

and

inositol

independently of any

was

apparently

response

monophosphate marked

of

sustained

platelets

levels

inositol

(vis ~ vis the

to

some agonists

significantly increased

phosphomonoesterase

activity

(i.e. levels of Ins-P2 and Ins-P~ were sustained with prolonged 30 min)

exposure to endothelin).

Whatever the substrate source for diacylglycerol, is the

(20-

only well characterized,

natural activator of protein kinase

C (8,9). Thus induction of its formation by agreement

with

reversibility

this metabolite

of

the

endothelin is

physiological

endothelin on isolated vessels by H-7, a protein

of

kinase C inhibitor

(5). Protein kinase C has been proposed to participate

1366

quite in

effects

in regulation

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the sustained phase of agonist-induced (9),

and

indeed

this

phase

contracted with endothelin of

this

peptide

difficult to VSMC

this

on

(i).

Also, the

isolated

'wash out' phenomenon

from binding

been

of

sustained

inositol

agonist-receptor metabolic events

support such

coupling, should

be

very

studies on cultured

its receptor

trisphosphate

decay of diacylglycerol might

effects

attributed to the extremely tight

association between endothelin and tions

vasoconstrictive

vessels are characteristically

(i) and has

smooth muscle contraction

is particularly prolonged in vessels

although

a notion

other

considered.

(i0). Our observa-

accumulation and slow of protracted

cellular

Indeed

we

autocrine

have obtained

preliminary evidence to indicate that secondary processes related to eicosanoid metabolism may be involved in of endothelin We

mediating vascular effects

(18).

conclude

that

phospholipase

C

mediated

phosphoinositide

hydrolysis constitutes

an important mechanism of signal transduction

for endothelin-induced

vasoconstriction.

ACKNOWLEDGMENTS:

The

authors

acknowledge

Ursula

Baur and Maria

B~rgin for technial assistance and Amanda de Sola Pinto for preparation

of

the

manuscript.

This

study

was

supported by the Swiss

National Foundation No. 3.827.087.

REFERENCES I. 2. 3. 4. 5.

6. 7. 8. 9.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10. Hirata, Y., Yoshimi, H., Takata, S., Watanabe, T.x., Kumagai, S., Kakajima, K., and Sakakibura, S. (1988) Biochem. Biophys. Res. commun. 154, 868-875. ii. Jones, P.A., Scott-Burden, T., and Gevers, W. (1979) Proc. Natl. Acad. Sci. 76, 353-357. 12. Scott-Burden, T., Bogenmann, E., and Jones, P.A. (1986) Exptl. Cell Res. 156, 527-535. 13. Scott-burden, T., Murray, E., Diehl, T., and Gevers, W. (1983) Hoppe-Seylers Z. Physiol. Chem. 364, 61-70. 14. Shayman, J.A:, and Morrison, A.R. (1985) J. Clin. Invest. 76, 978-974. 15. Duggleby, R.G. (1981) Anal. Biochem. ii0, 9-18. 16. Berridge, M.J. (1984) Biochem. J. 220, 345-360. 17. Rittenhouse-Simmons, S. (1979) J. Clin. Invest. 63, 580-587. 18. Resink, T.J., Scott-Burden, T., and B~hler, F.R. (1988) Biochem. Biophys. Res. Commun. (manuscript submitted).

1368