Vol. 183, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

March 16, 1992

Pages

864-871

THROMBIN-INDUCED CALCIUM OSCILLATION IN HUMAN PLATELETS AND MEG-01, A MEGAKARYOBLASTIC LEUKEMIA CELL LINE Yukio Yoshiaki

Department Medical * **

Ozaki, Yutaka Yatomi, Shinichi Wakasugi*, Shirasawa*, Hidehiko Saito**, and Shoji Kume

of Clinical and Laboratory Medicine, Yamanashi College, Tamaho, Nakakoma, Yamanashi 409-38, Japan

Kowa Research

Kannondai,

Institute, Kowa Co. Ltd., Ibaragi 305, Japan

First Department Nagoya University,

of Internal Tsurumaicho,

Medicine, Showaku,

School of Nagoya 466,

Tukuba, Medicine, Japan

Received November 19, 1991 SUMMARY:Digital imaging microscopy revealed that human platelets show periodic intracellular Ca++ elevation in response to 0.01 U/ml thrombin. MEG-01, a megakaryoblastic leukemia cell line, also responded with oscillatory intracellular Ca++ elevation (0.7-l times/min) to thrombin (O.OOl-O.O03U/ml). Cat+ transients appears to be fused with higher thrombin doses. With extracellular Cat+ concentrations of 0.1 mM or less, Ca++ oscillation could not be elicited, or even when present, it disappeared after a few spikings of [Ca”li. Extracellular Ca++ concentrations of 0.3 mM or more were required to facilitate ongoing Cat+ oscillation, suggesting an important role of Ca++ influx for Cat+ oscillation. 0 1992ricadem1c Press,Inc.

Cells

respond

to

the

intracellular

increase

in

Elevated

[Ca”]i

cellular excitable which

is

range Cat+

now known to play

processes. cells

a wide

such

The as

show biorhythmicity.

Ca++

of

Recently,

with

concentration a major

signals

sinoatrial

stimulators

cells there

([Ca++]i).

role

often

in

a number of

oscillate

and parabolic is

an

growing

with neurons evidence

ABBREVIATIONS: ACD solution, 15% acid citrate-dextrose solution; intracellular Ca++ concentration; EGTA, ethyleneglycol [Ca++li, ,N’-tetraacetic acid; HEPES, Nbis(m-aminoethyl ether)-N,N,N’ 2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid. 0006-291x/92 $1.50 Copyright 0 I992 by Academic Press, Inc. All rights of reproduction in arzy form reserved.

864

Vol.

183,

that

No.

cells

may

in

to

various

Cat+

have

(6). thrombin. cellular

not

been

presented

to

the

and

retains

response

has

been In

lets

AND

non-excitable

[Ca++]i for

BIOCHEMICAL

2, 1992

present

MEG-01, a

show

show

of

this

we have

while

sought

The several

that cell

[Ca++li to

oscillation

in

mechanism proposals

(2-4). human line

similar

determine Ca+*

fluctuations

demonstrated

responses

to to

phenomenon

of

COMMUNICATIONS

(1).

leukemia

fluctuation

contributes

periodic

elucidated,

functional

regular

RESEARCH

stimulators

a megakaryoblastic

We also Ca++

also

explain

study,

number a

fully

BIOPHYSICAL

to in

what

(5)

platewhich

platelets

response extent

observed

to extra-

with

MEG-

01.

MATERIALS

AND METHODS

Materials: Fura 2-acetoxymethyl ester and pluronic F-127 were obtained from Molecular Probes (Junction City, OR). All other agents were of analytical grade or better. Unless otherwise stated, cells were suspended in a HEPES buffer containing 145 mM NaCl, 5 mM, KCl, 1 mM CaCl 10 mM glucose, 0.1% bovine serum albumin, 10 mM HEPES (pH 7.4 7: Preparation of fura Z-loaded MEG-01 cells: MEG-01 cells were added into a hand-made silicone well glued to a cover glass. The upper surface of the cover glass had been pretreated with 1 mg/ml fibronectin to facilitate cell attachment. After 3 hours of incubation at 37” C, a considerable ratio of cells spread onto the the buffer was carefully withdrawn, and a mixed surface. Then, solution of 10 PM fura 2-acetoxymethyl ester, 0.01% pluronic F127, and 1.5% fetal calf serum was added into the well. After incubation at 37” C for 30 min, fura 2-acetoxymethyl ester that had not been taken up by the cells were washed out thoroughly by irrigating the well with the warmed HEPES buffer. Preparation of fura 2-loaded platelets: Human platelets were loaded with fura 2 as described previously (7). The fura 2loaded platelets were added into the silicone well described above, and left for 30 min until the cells were attached to the surface. Digital imaging microscopy: Digital imaging analysis of fura 2loaded cells was performed according to the method described by Williams et al. (8) with some modifications. Cells attached to a fibronectin-treated coverglass were placed onto a thermostatted microscope table, and were irrigated with a HEPES buffer or a thrombin solution which had been heated to 37” C. Fura 2 fluorescence was excited alternately at 340 nm and 380 nm with an emission wavelength of 510 nm. Video image was acquired with a silicon-intensified target camera (Hamamatsu, Japan), and analyzed with an image processor (PIAS LA-500, Japan). [Ca++li was calibrated using the 340nm/380 nm ratio. 865

Vol.

183, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION 1.

Ca++ oscillation

[Ca”]i

in human platelets:

was observed

Upon stimulation a periodic [Ca”]

i

single

with

change

in

fluctuation

changes two

with

in

a cell

human platelets

0.01

U/ml

fura

[Ca++ Ii

cell,

and a regular

thrombin,

la).

depicted

successive

at

peaks

in

The Fig.

varied

oscillatory

a resting

that

time lb.

from

change

showed

represented

course

of

[Ca”]

The interval 30 set

change

in

to

3 min

[Ca”]i

i

between in

a

as seen

b

Figure la. Digital imaging of [Ca”] i changes in human platelets activated with 0.01 U/ml thrombin. The ratio of fura 2 fluorescence was presented in colors (black to yellow in the increasing order of intensity). Figure lb. Time course of [Ca”li changes in human platelets (Left lower cell in Fig. la).

866

in

state.

human platelets

2 fluorescence,

(Fig. is

No oscillatory

Vol.

183,

No.

in

other

It

is

2, 1992

cell also

greatly

BIOCHEMICAL

types to

be

while

appeared

noted

differed

fluctuation

that

among

[Ca+*]i

were

evaluated

picture

in

with

of

all

had

the

[Cat+

Ii

in

cells.

to

stimulation

elevated

platelets

may

conventional

platelets.

Some

Our

showed

[Ca++]i

[Ca”li

levels,

findings

suggest

be sometimes

methods

since

COMMUNICATIONS

human

response

inert.

platelets,

RESEARCH

be absent

persistently

human

the

to

the

totally

responses

BIOPHYSICAL

individual

and another others

if

(1)

AND

they

that

give

actually

that

misleading the

average

behave

so dif-

ferently. 2.

]Ca++ Ii

Ca++:

elevation

Thrombin

in

approximately

of

thrombin,

increased

and

larger. 0.01

Almost U/ml

In

the

transient

in

MEG-01

cells

as low

as 0.001

U/ml

20% of

the

the

number

the

magnitude

all

the

of

often

range

0.001

U/ml

(Fig

2a and 2b).

a

0.001

U/ml

reached and 0.003

[Cat+ Ii In the

i

at

[Ca”]i

tended

rise

1 ,uM

in

to

be

response

to

maximum.

the

thrombin

U/ml,

presence

elevation

concentrations

elevation

with

i

elevated

a frequency of

elicited of

0.7-l

1 mM extracellular

CCa**li (PM)

b

t

i-Tin

Figure 2a. Ca*+ oscillation Fura 2-loaded MEG-01 cells

with

]Ca++li

[ca2+], (PM)

t

cells

[Cat+]

higher

showed [Ca”]

which

of

With

the

cells

oscillations

times/min

of

1 mM extracellular

induced

cells.

thrombin, of

with

0.003

U/ml

l-z”

induced by 0.001 U/ml thrombin. were first perfused with a buffer

containing 1 mM Ca+* without thrombin, and at the time indicated by the arrow, the perfusate was changed to a buffer containing 1 mM Ca++ and 0.001 U/ml thrombin. The trace is representative of 3 experiments. Figure 2b. Ca+* oscillation induced by 0.003 U/ml thrombin. Same as Fig. 2a, except that MEG-01 cells were stimulated with 0.003 U/ml thrombin, starting at the arrow. 867

Vol.

183, No. 2, 1992

3

BIOCHEMICAL

. . . . . . . f.

0 i= 2*

. . . . if

AND BIOPHYSICAL

. . . . i . . . . . . . j . . . . . . . i- . . . . . . i . . . . . . . j..

. . . . i-

. . . . . . i . . . . . _.I . . . . . I

/ I . . +.

I A._

.,.......

jkk$ip*

I

1 E

I

( /

/ .A..

...

I ..: . . . . . . . I . . . . . . A __.....

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)

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;

I

; I

I ,

/

0.001

;_

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1

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i ..__.

;

I

1

I

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I

~

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I

:

0.003

U/ml

I

RESEARCH COMMUNICATIONS

I 2.

... ... . I

/ -0.1

0.01 UYml! 1 min

U/ml

Figure 3. Thrombin dose-related changes in [Ca++]i elevation. MEG-01 cells were activated with 0.001 U/ml thrombin for the first 5 min, and then the dose was increased to 0.003 U/ml, and to 0.01 U/ml in a 5 min interval. The trace is representative of 3 experiments.

oscillation

Cat+,

thrombin dual

was

induced

a fused

[Ca++ Ii

(Fig.

unable

of

is

in

perfused

with

reduced lation

the

were

minutes

the

role

a buffer

upon the

With

0.1

ued

for

of Cat+ with of

Cat+

cells

for

0.001

U/ml

the

of

frequency of

of

stimulation,

(9).

when the

of (data

then

the

thrombin not

Fig.

the

did

shown). 868

and

then

thrombin [Ca”]i

of

and

oscil-

[Ca”]i

with reap-

extracellular

fluid.

oscillation

contin-

away

minimize

Since

several

were perfused

waned not

of

4a,

transient

gradually

for

Cat+,

Spiking

1 mM Cat+ to

Cat+,

and

oscillations,

thrombin

cells

no Cat+.

of

[Ca+‘]i

same dose

As shown in

virtually

mM extracellular

extracellular

elevation

the

intensity

influx

containing

addition

concentrations

each indivi-

1 mM extracellular

whether

of

of

concentrations

1 mM extracellular

disappeared

a while,

of

range

Ca++ on oscillation:

presence

containing

higher

presence

types

Ca++ concentrations.

a buffer

for

a sustained

by the

other

stimulated

immediately

peared

modified

for

slightly

showed the

limited

differed

determine

extracellular

To evaluate cells

in

to

an extremely

at

which

Thus,

fluctuation

Effect

Higher

slightly

pattern

as has been reported

the

which

and thrombin

3).

we were

[Ca++]i

3.

in

concentrations

MEG-01 cells,

Cat+,

observed

(Fig. the

extracellular

need

4b). for Cat+

Vol.

183,

No.

2, 1992

BIOCHEMICAL

0.001

AND

BIOPHYSICAL

U/ml

RESEARCH

COMMUNICATIONS

1%”

b

[ca**lt

(PM)

--O.=

0.001

U/ml 1%”

Figure by 0.001 Figure containing indicated buffer arrow, buffer. Figure buffer thrombin.

4.

Effect of Ca*+ depletion on Ca++ oscillation induced U/ml thrombin. 4a. MEG-01 cells were first perfused with a buffer 0.001 U/ml thrombin and 1 mM Ca*+, and at the time by the closed arrow, the perfusate was changed to a containing 0.001 U/ml thromibn and 1 MM EGTA. At the open the cells were again perfused with a Ca++-containing The trace is representative of 3 experiments. 4b. Essentially the same as Fig. 4a, except that the starting at the arrow contained 0.1 mM Ca*+ and 0.001 U/ml

contributes

as

evidence ion

for

of

[Ca++

cellular

an ]i

accord on

for

a

Ca++

There

0.1

Ca++,

lation,

include

into

this

thrombin

of issue,

report with

on

we next with

various

the

cell

on the

Ca++ release 4,

the

continuatof

11).

response

extracellular

extra-

appears

findings

contributes

C (2,

provide

concentration

necessity

a different

examined

for

Our

the

Ca++-induced

findings

oscillation

mM.

Ca++ influx

our influx

proposals

several

or

Cat+ minimum

mM and 0.3

phospholipase

stimulation

of

influx,

[Ca”li

oscillation been

Ca++

The

facilitating

extracellular

potentiation

role

previous

have

which

for

important

between with

Ca++

source

oscillation.

Ca++

somewhere

a

are

to in

of

extracellular

type

(10).

mechanism to

by

[Ca+‘]i

and To have

be good

which oscil-

Ca*+-induced an insight

of MEG-01 cells Ca++ concentra-

to

Vol.

183,

No.

2, 1992

BIOCHEMICAL

0.001

AND

BIOPHYSICAL

RESEARCH

U/ml

COMMUNICATIONS

lmi”

Figure 5. Effect of extracellular Cat+ on [Ca+*]i elevation induced by 0.001 U/ml tbrombin. MEG-01 cells were stimulated with 0.001 U/ml thrombin in the presence of 0.3 mM extracellular Ca++. Each of the thick and thin lines represents the (Cat+ ]i time course of a different cell.

tions.

MEG-01

0.001

U/ml

0.1

however, [Ca”]

i which

These

findings

for

interval

of

the

that

process

in

karyoblastic

is

for

feedback [Ca++]i

may be operative other

in

of

oscillation

[Ca++ Ii

leukemia

cell

is

Berridge

2.

Irvine

MJ (19901 RF

(1990)

geneis

provide

(4,

111,

]Ca++ ]i

one

of

has

the been

and we suggest

oscillation is plays

also

in MEGpossible.

an important

in MEG-01 cells,

J Biol Chem 265:9583-9586 FEBS Lett 263:5-g

C

induces

which

line.

870

5).

U/ml

to

indeed

REFERENCES 1.

the

trisphosphate,

Ca++ influx

that

of

on phospholipase

mechanisms

we suggest

Cat+,

(Fig.

for

appear

Cat+

system

evidence

a buffer

as 0.001

oscillation,

of

of

as low

inositol

of

lines

of

to

oscillations

required

findings

feedback

involvement

induction

our

Thus,

several

conclusion,

Ca*+ influx

with

a few minutes

peak when thrombin

This

by

role

that

production

supported

In

the

response

mM extracellular

in

a positive

the

0.3

occurred

proposed

01 while

With

Ca++.

perfused

transient

Ca++ mobilization.

this

were

in

elicited

for

mechanisms

cells

elevation

thrombin

suggest

with

]i

[Cat+

U/ml

stimulation.

evidence which,

less

a [Ca+*] i

of

no

when the

mM or

0.001

ration

showed

thrombin

containing

used

cells

a mega-

Vol.

3. 4. 5. 6. 7. 8. 9.

183,

No.

2, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Peterson OH, Wakui M (1990) J Membrane Biol 118:93-105 Harootunian AT, Kao JPY, Paranjape S, Tsien RY (1991) Science 251:75-78 Watanabe T, Yatomi Y, Sunaga S, Miki A, Nakao A, Higashihara H, Kurokawa K, Shimizu T (1991) M, Seyama Y, Ogura M, Saito Blood 78:2328-2336 Ogura M, Morishima Y, Okumura M, Hotta T, Takamoto S, Ohno R, Hirabayashi N, Nagura H, Saito H (1988) Blood 72:49-60 Ozaki Y, Yatomi Y, Kariya T, Kume S (1988) Biochim Biophys Acta 1012:87-96 Williams DA, Fogarty KE, Tsien RY, Fay FS (1985) Nature 318:558-561 Rooney TA. Sass EJ, Thomas AP (1989) J Biol Chem 264:1713117141

10. 11.

Yule Meyer

DI, Gallacher DV L (1988) T, Stryer

(1988) Proc

871

FEBS Lett 239:358-362 Nat1 Acad Sci USA 85:5051-5055