Inositol trisphosphate and thapsigargin discriminate endoplasmic reticulum stores of calcium in rat brain

Vol.

172,

No.

October

30,

BIOCHEMICAL

2, 1990

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

INOSITOL

TRISPHOSPHATE RETICULUM

'The

Johns

Pharmacology 725 N. 2Department University

Biological

Chemistry, Davis,

California,

of Clinical Rigshospititalet, DK-2100

Danish School of 2, Universitetsparken,

September

18,

R.

ENDOPLASMIC

Hanley2, and

Medicine Psychiatry 21205

School California

of Medicine Y5616-8635

Chemistry,

University Blegdamsvej Copenhagen, Denmark

Pharmacy,

811-816

Hospital Y

Department of DK-2100 Copenhagen,

Organic Chemistry Denmark

1990

Ca2+ accumulation into oxalate-loaded rat brain microsomes by thapsigargin with an IC,, of 2 nM and maximal inhibition 15% of the total A23187-releasable microsomal calcium store is to thapsigargin concentrations up to 100 pM. Inositol1,4,5-trisphosphate (IP$ maximally inhibits 40% of the net Ca2+ accumulation by whole brain microsomes. Its effects are non-additive with thapsigargin suggesting that the IP,-sensitive Ca2+ pool is a subset of the thapsigargin sensitive Ca2+ pool. Marked regional differences occur in Ca2+ transport rates and sensitivity to both thapsigargin and IP,. 'I' 1990 Acadrmlc press, 1°C. is at

ATP potently 10 nM.

DISCRIMINATE IN RAT BRAIN

Hopkins University School of Departments of Neuroscience, and Molecular Sciences, and Wolfe Street, Baltimore, Maryland of of

3Department

4Royal

AND THAPSIGARGIN STORES OF CALCIUM

David J. Hirsch', Michael Verma', Ole Thastrup3, S. Brogger Christensen4 Solomon H. Snyder'*

Ajay

Received

AND

1990

dependent inhibited Approximately insensitive

Thapsigargin classified cell

types

this

occurs

a highly

the

a non-TPA

by

discharging does

whom

Abbreviations: thapsigargin. Tg.

promoter

involve

of

all

the

cell

of

ER stores

more

potent

inositol,

be

The

areas

such

by

which

(2,3), Ca"-ATPase are

as

but (4).

heterogeneous cerebellum

appears

to

is

unclear

It

is

different

mechanism

(ER) which

IP, (I).

of

polyphosphates

(5),

tested

Ca'+-pool

(1).

reticulum

in

lactone

a variety

inositol

from

types

should

Ca2+

endoplasmic

Tg-sensitive

correspondence

sesquiterpene

Tg activates

stored

the

substantially

In

IP,,

(1).

generation

selectively

IP,

of

occurring

intracellular

Ca2'

with

all

naturally

inhibition

(6,7).

subcompartment

a tumor

not

releases

brain,

thalamus

*TO

as

selective IP,

in

(Tg),

influence whether

than a Tg

addressed.

1,4,5-trisphosphate;

ER,

endoplasmic

reticulum;

0006-291)(/90 811

$1.50

Copyright 0 1990 by Academic Press. Inc,. All rights of reprodrrction iIt any form reserved.

Vol.

172,

No.

recruits have

2, 1990

Ca"

from

examined

We show

that

sensitive

all

ER or

the

influence

IP,

and

pool

variations

BIOCHEMICAL

is

from of

Tg

only Tg

of

the

BIOPHYSICAL

IP,

on

only

the

Tg

a

In

45Ca2'

flux

portion

sensitive

the

in

rat

of

ER

Ca'+

of

Ca"

pool

COMMUNICATIONS

present

study

brain

microsomes.

and

with

we

that

the

marked

IP,

regional

brain.

MATERIALS

AND

METHODS

45(Ta2+ was purchased from NEN-DuPont. Thapsigargin was isolated as described were of the highest purity available as

Materials. Calbiochem. chemicals

RESEARCH

a subpopulation.

and

influence

a subset

throughout

AND

IP, previously reported

was

obtained (8). All previously.

from other

sCa2' Uptake Assay. 45Ca" uptake was studied in 50 mM KCL, 50 IJIM K, oxalate, 10 mM Hepes, pH 7.5, 3% polyethyeleneglycol, 2 mM MgCl,, 20 units/ml creatine phosphokinase, 100 PM total CaCl,, 0.1 &i/ml 45Ca", 2 ml? DTT and indicated additions. Free calcium concentration was adjusted to 0.1 pM with HEEDTA using a Ca2+ sensitive electrode (Orion). Microsomes from whole brain, unless otherwise indicated, prepared as previously described (6), were added to a final concentration of 50 pg/ml to initiate 4'Ca2+ uptake. Incubations were performed at 37°C in a total volume of 0.5 ml for indicated times and the assays were terminated by rapid filtration using polyethyleneimine coated 0.45 pm Millipore filters. The filters were washed twice with 3 ml of ice-cold wash buffer containing 100 mM KCl. 25 mM KL oxalate, 10 mM Hepes, pH 1.0, 3% polyethyleneglycol and 2 mM EDTA. Radioactivity was measured in 5 ml Redi-solv scintillation cocktail (Beckman) using a Beckman LS-38 beta counter. Protein Assay. acid protein

assay

Protein concentrations reagent (Pierce)

were determined bovine serum

with

usirlg albumin

the as

bicinchoninic a standard.

rat

brain

RESULTS To which

label

ER

should

have

subcellular of

- 1 pM

in

only by

vanadate,

an

10

pM

of

mitochondria

ruthenium

which

45Ca2'

in

in

assay (data

is

maximal the

linear

microsomes

and

PM

sensitive

of IP, (Figure

Ca"

ER Ca2'

ER

CaJt

is

evidenced .shown)

for

about at

or

pumps

(10).

not

(Fig.

not

of

an hour 45

37°C with min

be

We also

vesicle

of

uptake

between 1).

45Ca2t

by

5 mM bJ

include

50 but

mM not

accumulation

accumulation

a plateau (Figure

the

Ca"

inhibitors

vesicles

membrane

all

unaffected all

ER

a Km system

abolished is

into

'IsCazi

has

conditions

oligomycin,

insensitivity At

can

shown).

plasma

pump uptake

accumulation

pg/ml

accumulating

ER Ca"

these

accumulation

Ca2+

by

about

10

(data

Lack

the

1)

45Ca2t

microsomes,

Ca2+

mitochondrial

Under

pump.

supports

the

uptake.

azide,

major

because

Km of

preparations the

accumulation

absence Tg

PM 4'Ca2t.

-10

preparations

digitonin

microsomes

0.1

the

utilize

another

to

selectively

our

mitochondria,

1 mM sodium not

we

We employ

of

but

membrane

selectively,

nlicrosomal

red,

plasma

half

the

Ca"

few

ATP

inhibitor

oxalate,

PM

very

contrast

We measure

accumulation

of

of

component.

0.1

(9).

stores

process

to

by

brain

rat

PO and uptake

180 is

min

50

and

abolished

ATP. differ

in 21.

the Tg

extent potently

to

which inhibits

812

they

impair

accumulntiorl

.IsCa'+

accumulation with

maximal

by 858

Vol.

172,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

300

,’

,

-CONTROL

L”; : r -ATP

1

0

60

TIME

Time cc~urse presence and

the

inhibition

Half

at

maximal

range

of

various

10

nM

potencies

by Tgis

and

inhibition

further

reported

(1)

for

and

in

Tg

in

by

Tg

increasing

inhibiting

with

was

occurs

at

2 nM, in

about levels

ATPase

measurrd

il;

up to 100 PM.

cytoplasmic

the ER Ca"

approximatelymonophasic

Accumulation three times.

at concentrations

of '15Caz+ accumulation

(4).

of

The

a calculated pseudo-Hill

the

Ca"

in

inhibition coefficient

-1.04. The Ca2+ ionophore

half

(min)

into rat brain microsomes. The experiment was replicated

no

inhibition

systems

curve of

of Ca accumulation absence of ATP.

0 180

120

maximal

accumulation displays calcium

A23187

inhibition

at about

by A23187

is

a pseudo-Hill permeability

inhibits

50 - 80 nM.

steeper

than

coefficient

of

of vesicular

12

100% of 45Ca2+ accumulation for -1.45

The slope Tg.

for

The A23187

consistent

with

at 1 PM with

inhibition

of 45Ca2+

sensitive its

process

increasing

the

for The

in

membranes.

10

8

6

4

- [LOG1 W) Figure

2. Inhibition of calcium accumulation. presence of indicated amounts of thapsigargin replicated three times.

Calcium accumulation was assayed (TG), A23187 (.423), or IP,.

813

60 min experiment

the wils

Vol.

172,

No.

2, 1990

BIOCHEMICAL

Pharmacologic

Sensitivity

of

AND

Rat

BIOPHYSICAL

Table

1

Brain

Microsomal

RESEARCH

Calcium '%a'+

8 Control

pM IP, IP,

pM nM

+ LOO pg/mL

62.0 100.0 11.5 11.9 11.5 0.15

heparin

TG

nM TG + LO pM IP, nM TG + 100 pg/ml heparin nM TG + 10 pM A23187

accumulation was assayed of agents. Values are means

as described

Calcium

amounts performed

in

inhibits

1 - 10 PM and half

shallow

with To

of

to block

these

IP,

(10

nM)

is not

influenced

apparent

to its

1).

at

separate

indicated

with

experiments

receptors

when

by 40% with

80 nM.

of about IP,, and

The curve sensitive

by 100 pg/ml

indicating

that

is

resistant

to

in

the

IP,

pools,

is

fairly

of heparin,

which

release 90%.

combined.

The

acting

is known

of Ca"

by almost

Tg is not maximal

we examined

by 10 PM IP, of net

and associated

45Ca2+ accumulation

10 nM Tg and 10 PM IP, are

which

for

40% inhibition

(11)

effects

maximal

-0.4

Tg

Maximal

reversed

inhibits

studies

in 45Ca2t release

variations

in sensitivity

in various

brain

with

60 min

five

(12).

No further effect

of Tg

upon IP, receptors.

concentrations

of

Tg,

is

by 10 pM A23187.

Preliminary differences

(Table

by heparin

accumulation

abolished

of

is completely

binding

is

effects

coefficient

agents

maximally

effect

%a'+

maximal

relationships

'5Ca2f accumulation

Tg

3.2 4.0 1.1 0.7 1.0 0.1

for of

45Ca2+ accumulation

net

a pseudo-Hill

evaluate

mixtures

2 S.E.M.

k + f + ? f

duplicate.

IP, maximally at

Accumulation

Accumulation

Treatment 10 10 10 10 10 10

COMMUNICATIONS

highest

Regional Brain Region Whole Brain Cerebellum Striatum Bulb ThaLamus/Midbrain Spinal Cord

Olfactory

"Ca'+ accumulation IIM Tg or LO WM IP,. "Indicates statistically

Net

in

brain

of oxalate

microsomes

by

to IP, of “5Ca2' accumulation

regions

levels

from

absence

(Table the

2).

Total

Variations

in

Rat

Table Brain

+ k ? + k -f

was assayed ALL values are significant

IP,

We now

(6).

in

double

2 Microsomal

the presence

the

Ca"

next

regional show

varies highest

marked

of oxalate markedly levels

in

Accumulation % Sensitive

Ca2+ Accumulation (nmol/ms protein) 119.5 212.4 117.8 82.8 54.7 49.8

apparent

ER 45Ca2+ accumulation

almost

cerebellum,

revealed

T,?

5.3 4.2 4.3 7.6 4.8 5.3

87.0 92.5 86.8 90.0 85.2 85.0

as described means -t S.E.M. differences

814

2 + k k 2 -t

IP3 0.8 0.5 1.8* 0.7x" 0.5* L.L*

37.0 50.7 35.2 8.1 12.2 5.1

for 60 min in the presence or of 6 8 ex-periments performed vs. cerebellum. p < 0.001.

f 2 + 2 -t ?

0.6 3.8 4.7* 4.1x" 3.7x" 2.9*

absence in

of

duplicate.

100

Vol.

172,

the

No.

BIOCHEMICAL

2, 1990

striatum

followed

thalamus/midbrain Effects ‘IScaL+

inhibits microsomes. 5

of

IP,,

and

Tg

olfactory

in

is

the

most

about

as

In

the

cord

quarter

and

the

of 10

inhibition

cord

and

that

cerebellum

less

accumulation are

b-y

regional

sensitive,

as

spinal

in

PM

IP,

striatsl

olfsctorv

bulb

only

If,.

great

in

more

than

differences while

sensitive.

twice

a

, spinal

there

less

about

somewhat

hv

'sCa'+

being are

accumulation as

inhibits

bulb

with

COMMUNICATIONS

The

regionally.

blocked

However.

thelamus/midbrain

onlg

vary

RESEARCH

bulb.

thnlomus/midbrain is

esamined.

olfactory

50%

accumulation

Lfnlike regions

IP,

accumulation in

BIOPHYSICAL

accumulation

of

while

- 128

the

by

display

cerebellum.

AND

the

5'0%

with

spinal

The

amount

of

the

spinal

cord

in the

cord, Tg

all

cerebellum striatum

resistant

and

brain

and ER

"Ca"

thalarnus/midbrain

cerebellum.

DISCUSSION The are

main

finding

of

heterogeneous.

ATPase, of

Tg,

maximally

discrete of

regions

with

in

cerebellum

the

and

by

ER pools Ca"

55% of

is

Ca"

to

between Tg

in by

the

varies

the

the

Ca"

brain.

Tg

in

brain

existence

among

smaLlest

pools

the

the

in

Tg

the

resistant

Ca'+

suggesting

ATPases

sensitive

variation largest

oi

accumulation

ER Ca".

ER

which

the

the

inhibiting

resistant

a two-fold rind

that

about

accumulation

about

is

acts only

sensitive

Ca"

study

which

influences Tg

proportion

this

The differeilt

resistant

pool

spinai

cord

and

thalamus/midbrain. IPj maximal The

influences inhibition

by

IP, , as

b.y

other

which

of IP,

are

to

influences

the

cerebellum

IP,.

of

Tg.

Tg

bulb The

the

in

of

non-mitochondrial microsomal greatest receptors

(7).

affected

in

highly

sensitive

rat

There to

HOW these

IP,

and

to greater

show

close with of

815

is

of

ER pools

Ca"

accumulation in

IP,

the

Ca“ are

regional

effect

on

Ca" Tg

thalamus/midbrain.

but

For

we

By

show the

now

response

effects

of

also

than

to

in

observe

with

regions

with

densities relate

IF,

variations

of

greatest

contrast,

little

regional

correspondence the

of

Similarly,

marked

receptors

ER

observed

only

fashion. Tg

with

from

also

greater

or

Ca"

as

evident

for

which

those

localizations

on

intermediate

brain

a

Tg

cord

(i), is

(7).

Tg

preparations.

thalamus/midhrain.

is

accumulation

preparations. sensitivity

is

in

and

spinal

variations

release

IP.

thaII

brain

the

a S - 8 fold or

the

whole

Ca"

rates

and

IP,

are

studies ‘pa*+

in

ER

influence

Tg

exerts cord

than

regional

Autoradiographic

to of

IP,

spinal

more

pools

appears

between IP;.

the

striatum

ER Ca"

extent

to

40%

reflects

effects

relationship

than

and

the

about

of

accumulation IP;

as

sensitivity

cerebellum

to

Tg,

portion

IP,

initial

(13.14).

the

IP,

by

affect

Another

olfactory

being

accumulation

not

sensitive

in

smaller

accumulation

Ca"

does

additive.

both

substantially

investigators

differences in

of

inhibition

not

a

of Tg

sensitive

IP,

Vol.

172,

No.

and

insensitive

Ca"

ATPases

to

Tg has

Tg

BIOCHEMICAL

Ca"

pumps

have

been

yet

been

enzymes

in

not

different between

2, 1990

sensitive

is

unclear.

molecularl,y

and

BIOPHYSICAL

Recently cloned

established. various

AND

several

(15,16).

Conceivably, brain

insensitive

regions Ca"

RESEARCH

different Their

reveal

intracellular

relative

measurements will

COMMUNICATIONS

sensitivity of

possible

mRNA

for

these

correlations

pools.

ACKNOh'LEDGMENTS

S.H.S., Company. 1987-1990. Foundation.

Supported by USPHS grant MH-18501, Research Scientist Award DA-00074 to Training Grant ES-07141 (A.V) and a gift of the Bristol-Myers-Squibb S.B.C. and O.T. supported bp the Danish State Biotechnology Program M.R.H. is the recipient of an award from the ILSI Research

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. LO. 11. 12. 13. 14. 15. 16.

Thastrup, 0. (1990) ATents and Actions 2, 8-15. Jackson, T.R., Patterson, S.I., Thastrup, 0. and Hanley, M.R. (1988) Biochem. J. 253, 81-86. Takemura, H., Hughes, A.R., Thastrup, 0. and Putney, J.W., Jr. (1989) JBioL. Chem. 264, 12266-12271. Thastrup, O., CuLlen, P.J., Drbak, B.K., Hanley, M.R. and Dawson, A.P. (1990) Proc. Natl. Acad. Sci. USA 87, 24662470. Berridge, M.J. and Irvine, R.F. (1989) Nature 341, 197-205. Supattapone, S., Danoff, S.K., Theibert, A., Joseph, S.K., Steiner, J. and Snyder, S.H. (1988) Proc. Natl. Acad. Sci. USA B. 8747-8750. Verma, A., Ross, C.A., Verma. D., Supattapone, S. and Snyder, S.H. (1990) m Regulation, in press. Rasmussen, U., Christensen, S.B. and Sandberg, F. (1978) Acta. Pharnra. Suet. 15, 133-140. Carafoli, E. (1987) Ann. Rev. Biochem. 56, 395-433. Gill, D.L., Chuch, S.H. andwhitlow. C.L. (1984) J. Biol. Chem. a,lO80710813. Worley, P.F., Baraban, J.M., Supattapone, S., Wilson. V.S. and Snyder, S.H. (1987) J. BioL. Chem. 262, 12132-12136. CulLen, P.J.. Comerfield, J.G. and Dawson. A.P. (1988) FEBS. Lett. 2-38, 57-59. Leslie, B.A., Burgess, G.M. and Putney, Jr.. J.W. (1988) Cell Calcium zj 9-16. Baumann, 0. and Walz, B. (1989) J. Corm. Physiol. 165, 627-636. (1989) J. Biol. Burk, S.E., Lytton, J., MacLennan. D.H. and ShuLl. G.E. Chem. 264, 18561.18568. Gutenski-Hamblin, A.M., Greeb, J. and Shull, G.E. (1988) J. Biol. Chem. 263, 15032-15040.

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