Inositol 1,4,5-trisphosphate-induced calcium release from platelet plasma membrane vesicles

BIOCHEMICAL

Vol. 150, No. 3, 1988

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

February 15, 1988

INOSITOL

1,4,5-TRISPHOSPHATE-INDUCED FROM PLATELET Appavoo

December

RELEASE

PLASMA MEMBRANE VESICLES

Rengasamy

Department University of Chicago, Received

CALCIUM

1021-1026

and Harold

Feinberg

of Pharmacology Illinois at Chicago, Illinois 60612

7, 1987

SUMMARY : A platelet membigg;2preparation, enriched in plasma membrane markers, took up in exchange for intravesicular it after the addition of inositol 1,4,5-trisNa+ and released phosphate (IP3). The possibilility that contaminating dense tubular membrane (DTS) vesicles contributed the Ca2+ released by IP3 was eliminated by the addition of vanadate to inhibit Ca+ATPase-mediated DTS Ca2+ sequestration and by the finding that plasma membrane vesicles exhibit Na+-dependent Ca2+ uptake. released by IP3 was dependent on low extravesicular Ca2+ concentrations. IP3-induced Ca2+ release was additive to that released by Na+ addition while GTP or polyethylene glycol (PEG) had no effect. These results strongly suggest that IP3 facilitates extracellular Ca2+ influx in addition to release from DTS 0 1988Academic mess, Inc. membranes.

In

many

production mediate

cell of

Ca2+

types 1~3

stimulus-response

which

release

acts

from

DTS membrane

vesicles

and releases

Ca2+ when IP3

ized and thus

platelets causes far

activated that

IP3

elevated presumably

triggers of

suggests

that

by IP3

(6,7).

cause

steady

level

by activating

Ca2+

(3,4).

Ca2+

release

Ca2+

influx

On the a greater in

granular

plasma

hand

several of

cation

(5).

platelet Mg2+ATP

stores Evidence

membrane studies

is

not

suggest

Ca2+ and

extracellular channels

to

permeabil-

internal

cytosolic of

of

saponin

from

presence

divalent

presence In

through

other

Human

contents

increase the

the

the

messenger

(1,2).

in

added

dense

involves

intracellular

stores

accumulate

secretion

agonists

as an

internal

is

coupling

an

Ca2+ in

platelet

0006291X/88 $1.50 1021

Copyright 0 1988 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Vol. 150, No. 3, 1988

plasma

BIOCHEMICAL

membranes

T-lymphocytes

(8-10).

indicate

can be activated IP3

in

that

transmembrane IP3

vesicles

had

been

ion

clamp

channels

influx

in

release

with

of

plasma

the

human

membrane

involvement

platelets.

from

loaded

studies in

We investigated

(11).

Ca2+

patch

Ca2+

Ca2+

induced that

Recently that

by IP3

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

We report

platelet 45Ca2+

of here

plasma

membrane

by Na+-Ca2+

exchange

mechanism.

MATERIALS

AND METHODS

Platelet plasma membrane was prepared as described previously (12,13). Briefly, fresh platelets were washed twice with Ca2+-free Ardlie buffer, pH 6.5 and sonicated in 25 mM Tris buffer, pH 7.4 containing 100 mM NaCl, 3 mM MgC12 and 50 uM leupeptin for 20 set (setting 6, Model W185, Heat Systems and Ultrasonics, Inc.). After centrifugation (1,500 x g for 15 min), the supernatant was fractionated on Percoll gradients at pH 7.4 and then at pH 9.6. The plasma membrane fraction was mixed with two volumes of 150 mM NaCl and 10 mM Tris-HCl, pH 7.4 and centrifuged at 200,000 x g for 1 hr. The membranes were washed twice and resuspended in 150 mM NaCl and 10 mM Tris-HCl, pH 7.4. Ca2+ uptake was measured by diluting a small volume (2 pl, 20 ng) of Na -loaded vesicles (incubated at 37OC for 30 mln) into 150 pl of a medium containing 10 mM Mops-Tris (pH 7.4, 37OC) with The reaction was stopped by 2 pM 45Ca2+ and 150 mM KC1 or NaCl. the addition of 200 pl ice-cold Mops-Tris buffer (5 mM, pH 7.4) The vesicles were filtered containing 150 mM KC1 and 5 mM LaC13. on Millipore membranes (0.45 pm) and washed twice with 5 mM Mopscontaining 150 mM KC1 and 2 mM LaCl3. Tris buffer (pH, 7.4) 45C,2+ remaining in the vesicles was determined with a liquid was calculated scintillation counter. Na+-dependent Ca2+ uptake by subtracting the uptake obtained in NaCl medium from the uptake Protein was determined according to by vesicles in KC1 medium. the method of Bradford (14).

RESULTS Previous of (13)

results

a

Na+-Ca2+

-

Fig.

membrane Ca2+ loaded

exchange la

vesicles

concentration. with

from

shows in

AND

our

DISCUSSIW

laboratory

mechanism the

time

in

Ca 2+ by means

platelet

course

KC1 and NaCl After

indicated

5 min,

medium

plasma

at

presence membrane

Ca2+ uptake

20 pM IP3

of Na +-dependent 1022

of

the

by plasma

2 pM extravesicular addition Ca2+ uptake

to

vesicles (i.e.

KC1

Vol. 150, No. 3, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

$u 0.5’0

1

Time (mid

2

3

Time

(mid

4

5

Fiaure la. Time course of Ca2+ uptake y platelet plasma membrane vesicles. A small volume (2 pl) i of plasma membrane vesicles loaded with 150 mN NaCl and 10 mM Tris-HCl, pIi 7.4 was equilibrated at 37OC for 30 min and diluted 75-fold into a medium containing 20 mMMops-Tris (pli 7.4,37OC), 2 pM 45 Ca2+ and 150 mM KC1 (*) or NaCl (0). Vesicles were filtered and Na+-dependent Ca2+ uptake ( 0 ) was determined as described in "Materials and

Methods".

Fiaure lb. IP3-induced Ca2' release from plasma membrane vesicles. Vesicles were Ca2+ loaded as described in Fig. la for 5 min at 37OC. Ca2+ release was initiated by adding IP3 (20 J.LM, o,A) or the same volume of vehicle (5 mMTris-HCl, pH 7.4, *, 0) to 150 pl of the assay medium containing KC1 (solid lines) or The reaction was stopped at the indicated NaCl (dotted lines). times and analysed for retained Ca2+ as described above.

medium) IP3

immediately

no further

released Ca2+ uptake

non-Na+-dependent less

Although it

is

tributed

to the

contaminating

by

uptake

exchange

tion system,

has

means

trapped

been

for shown

by DTS membranes however,

(15).

vanadate

(a) (b)

Ca2+ to

to

In have

Na+-Ca2+

inhibit

by a much

Ca2+ released

DTS

was

order

membrane

vesicles

for

this

accumulated activity

exchange, present

ATP-dependent

(100 pM) was added.

to

occur in

the

utilizing or

in

in con-

Ca2+

(c)

45Ca2+

DTS vesicles. Ca2+ seguestra-

No Mg2+ or ATP was added

1023

of

released

plasma

Ca2+ -ATPase

already

presence

2).

by IPS.

of

ATP,

of

contaminating

have

the

accumulated

The amount

predominantly

that

Ca2+ released

Ca2+ medium)

(Fig. is

possible

or

Vanadate

IPS.

preparation

endogenous in

of

DTS vesicles

instance

In

lb).

NaCl

(i.e.

concentration

our

origin,

first

addition

on IP3

(Fig.

was observed.

mechanism

Ca2+ by the

dependent

Ca2+

to

the

Na+-dependent

assay Ca2+

Vol.

150,

No.

3. 1988

BIOCHEMICAL

I P3

AND

BIOPHYSICAL

concentration

WI)

RESEARCH

COMMUNICATIONS

2. Concentration dependence of IP2-induced ca2+ release from platelet plasma membrane vesicles. Plasma membrane vesicles were diluted 75-fold into a medium containing 150 HIM Xl, 20 UIM Mops-Tris (pH, 7.4) and 2 PM 45Ca2+ at 37OC. After 5 min, Ca2+ release was initiated by the addition of varying concentrations of IP3. After incubation for an additional 5-mih vesicles were filtered and the Ca2+ retained was measured. Ca2+ released was determined by subtracting the amount of Ca2+ retained by IPgtreated vesicles from the Ca2+ content of control vesicles. Fiaure

uptake

was

release

was not

the

not

contrast

exhibit

pure loaded

Na+. up

released Thus,

Ca2+

by

Ca2+-Ca2+

exchange

membrane

DTS vesicles.

DTS membranes

released

about

Ca2+

5 min

in by

about

(13).

5% of

Ca2+

uptake

Ca2+

in

Thus,

it

involved

is in

or

could

when by

from

that

loading

50 rnM

have

Adunyah

unlikely

passive

for

not

Ca2+ release

Ca2+

relatively

exchange

mechanism.

Ca2$-induced

60% of

electrophoresis

DTS vesicles exchange

did

50 mN NaCl

However,

free-flow

ATP-dependent

of

is

of

our

vesicles

Na+-Ca2+

(16).

Ca2+ loading

excluded

membrane

the

membranes

from

vesicles,

Ca2+

we have

of

contaminating

lack

Thus

Addition

only

the

IP3-induced

mechanism.

the

demonstrated DTS

by

while

by IP3

membrane

obtained

Ca2+

released

exchange

membranes

diffusion

Ca2+

plasma

plasma

with

shown).

ATP-dependent

vesicular

DTS

not

(data

from

Na*-Ca2+

Na+-dependent

5%)

the

with

Ca2+-loaded

than

that

stemmed

In

(less

affected

possibility

vesicles

to

increased

taken

and

Dean

platelet

Na+-Ca2+

of

M'S

or

membrane

vesicles. The membrane

mechanism was

of

investigated.

IP3-induced Table 1024

Ca2+ 1 shows

release the

from amount

plasma of

Ca2+

Vol. 150, No. 3, 1988

BIOCHEMICAL

ca2+

TABLE 1.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of IP3-induced Ca2+ release plasma membrane vesicles

from

dependence

platelet

Free [Ca2+] in uptake medium Ca2+ uptake (nmol/mg) Ca2+ released by IP3 (nmol/mg)

(PM) 1.90

2.68 0.38

2.40

4.50

2.84 0.38

24.00 6.27 0.06

12.00 4.99

3.62 0.28

0.14

Plasma membrane vesicles (2 ~1) preloaded with 150 mM NaCl and 10 mM Tris-HCl, pH 7.4 were mixed with 75 vo'lumes of a medium containing 20 mM Mops-Tris (pIi 7.41, 150 mM KC1 and 45Ca2+ at indicated concentrations for 5 min at 37OC. IP3 (20 uM) was After 5 min the reaction was added to induce Ca2+ release. Ca2+ released by vesicles at stopped and vesicles were filtered. different concentrations of Ca2+ was determined as in Fig. 2. Free Ca2+ concentration was determined by a Ca2+ electrode.

uptake

increased

IP3-induced Ca2+.

Ca2+

This

PEG are Ca2+ addition

release agreement

known

to

cause

at

least

of not

several

reports

some

systems

GTP and

(4,17). of

(18).

IP3-induced However,

affect

platelet

IP3-induced

we have plasma

Ca2+ release Ca2+

(Fig.3).

from

3.

extravesicular

activation

shown

the

mechanism

that

induced

that

IPg-induced

membrane

Effect

from

released

of IP3 and other

the

GTP and plasma

by

IP3

and

Na+ were

of

IPg-induced

Ca2+

by Na+.

population

Calcium releose

Fiaure

low

of both

is different

predominantly

on

a significant

however

Ca2+,

5 pM GTP or 2.5% PEG or a combination

that

Thus

extravesicular

dependent

with

in

indicating

additive

in

was

in

membrane vesicles

release

a rise

is

release

PEG did

with

Ca2+

release

previously

from

loaded

with

condition

Ca2+ releasing

agents on Ca2+

release from plasma membrane vesicles. Plasma membrane vesicles were 45Ca2+ loaded in KC1 medium as described in Fig. 2 for 5 min at 37OC. Ca2+ release was initiated by the addition different agents at indicated concentrations 2.,5%, Na+-10 mM). At the end of minated and vesicles were filtered. was determined as in Fig. 2.

1025

(IP3-20

5 min the

pM, GTP-5 pM, PEG-

reaction

was ter-

The amount of Ca2+ released

a

Vol. 150, No. 3, 1988

Ca2+ by means other

cells,

inositide

of

salivary

exhibit

of

IP3

gland

and

cytosolic

Ca2+

in

and rat

direct by

Ca2+ uptake.

Platelets

receptor-mediated

in transmembrane provides

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Na +-dependent

turnover

in

here

BIOCHEMICAL

increase

cytosolic

Ca2+.

pituitary

cells

Ca2+ flux

(19-21).

evidence means

of

that a

The

platelets

IPg-induced

in

Studies suggest

like

many

phospho-

on Ca2+ the

involvement

finding may

flux

presented also

transmembrane

augment Ca2+

influx. ACKNOWLEDGEMENTS This work was supported by NIH grant Dr. Guy C. Le Breton for helpful discussions S. Mitchell for technical assistance.

HL 29721-03. We thank and A. Pettigrew and

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