Vol.

174,

No.

February

3, 1991

14,

Uptake

BIOCHEMICAL

RESEARCH

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of the InsPs-sensitive

smooth-muscle

Ludwig

Missiaen,

cells: Humbert

Department Received

BIOPHYSICAL

1991

characteristics

aortic

AND

December

different

K.U.Leuven,

Ca2+ pools

in porcine

of the Ca2+-uptake Ingrid

De Smedt, Guy Droogmans, and Rik Casteels

of Physiology,

17,

and -insensitive

C!a2+ sensitivity

1183-1188

B-3000

Declerck,

Leuven,

mechanism

Lutgart

Plessers

Belgium

1990

Summary. We have investigate the Ca2+-uptake characteristics of the InsPg-sensitive and pools in permeabilized cultured porcine aortic sm oth-muscle -insensitive pool, which was also GTP sen@ve, had a high Ca 2+ affinity cells. The InsP3-sensitive Ca and was highly oxalate permeabl 9+ The InsPg-insensitive Ca store, which was also GTP insensitive, had a much lower Ca affinity a d presented a low oxalate permeability. ‘l&e 0th pools decreased at high free [Ca tt+ 1, although these cells did not ha2v+ea Ca mechanism. This decreased loading of the InsP3-sensitive Ca pool 2tt must be taksp into consideration ~92 investigating a possible Ca rele se. Part of the Ca uptake into the InsP3-insensitive pool was not affected by the Ca I+ -pump inhibitors vanadate, thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone. ‘i’ 1391 RcadamlcPrfS.5, Inc.

Inositol

1,4,5trisphosphate

Ca2+ store [l].

Only

part

InsP3 [2,3], suggesting by their

(InsP3)

kinetics

mitochondrial [4] believe

of the Ca2+ accumulated

the existence of

Ca2+ that

Ca2+

mechanism

in

the InsP3-sensitive

both

pools

seems to be more sensitive [6,7]

and

(4)

may

change

a non-mitochondrial pool is released by

Ca2+ compartments

as a Ca2+/H+ BC3H-1 from

present [8]

distinguishable

chromaffin

inhibitors the

it

was Ca2+

which

Meyer

would that

sequestration

(1)

The

high

others assume that it is while that

the InsP3the uptake

InsP3-sensitive

acinar myogenic

Ca2+

pool.

to be associated molecular cells could

not be sensitive

Ca 2+

The

a totally

pool InsP3new

[9]. (3) The Ca2+

under

differentiation

(2)

with

weight

certain

to Ca2+-pump

to caffein-sensitive

non-

and Stryer

Ca2+ 2,5-di-(tert-butylj-1,4-benzohydroquinone

cells is claimed an unusually

observed

While

slowly,

InsP3-insensitive

Ca2+ pool in pancreatic

exchanger

InsP3-sensitive

with

-insensitive

the slow uptake kinetics, [S]. Evidence is accumulating

some differences: than

and

debated.

Ca2+ pool loads up rather

Ca2+ pump

myoblasts

InsPg-sensitive

cells is still

to the Ca2+ -pump

of the InsP3-sensitive In

from

heterogeneous

different

these

that presents very rapidly

thapsigargin

Ca2+ pool in bovine

type of intracellular pump

in

pools in agonist-stimulated

InsP3-insensitive Ca2+ pool would be loaded sensitive pool

function

Ca2+

in this

of at least two

accumulation

the

sensitive

intracellular

InsP3 sensitivity.

The

(tBuBHQ)

releases

conditions

inhibitors

induced

[lo].

a drastic

sequestration

[ll].

There was a concomitant switch from the non-muscle (SERCA 2b) to the muscle (SERCA 2a) isoform of the endoplasmic reticulum Ca’+-pump (De Smedt et al., unpublished results). This suggests, although it was not yet proven, that the 2 types of Ca ‘+-stores in BC3H-1 cells could

be filled

by a different

type of Ca2+-pump.

Vol.

174,

No.

BIOCHEMICAL

3, 1991

Because of these indications the

importance

homeostasis

of

in intact

InsP3-sensitive addressed

in

-insensitive

such

in favour

possible

cells,

AND

BIOPHYSICAL

of a different

differences

we have further

in

Ca 2i

investigated

RESEARCH

uptake uptake

mechanism, for

this

Ca2+ -stores in aortic smooth-muscle was whether the Ca 2+ sequestration

work

a different

Ca2+ sensitivity.

and because of

understanding

the Ca2+ -uptake

and -insensitive

Ca2+ pools presented

COMMUNICATIONS

the

characteristics

cells. The main by

the

of the

question

InsP3-sensitive

Because the binding

Ca2+ we and

of InsP3 to

the cerebellar receptor and hence its Ca 2+-releasing effect is impaired at high [Ca2+] [12-141, it was impossible to study the Ca 2+-dependency of the uptake mechanism of the InsP3-sensitiveand -insensitive Ca 2+ pool by comparing the 45Ca2+ uptake in the absence and presence of InsP3

in

loading

media

containing

various

free

[Ca2+].

We therefore

the pools, in the absence of InsP3, at different

medium

and replacing

cells were

challenged

Materials

and Methods

it with with

an efflux

InsP3 at this

medium low

developed

a technique

free [Ca2+], then

containing

100 nM

removing

[Ca2+].

of first

this uptake

Thereupon

the

[Ca2+].

Vascular smooth-muscle cells from thoracic pig aorta were isolated and cultured as described [15]. The cells were subcultured and used between the 5th and 12th passage. More than 98 % of the cells reacted with monoclonal anti-cu-smooth-muscle-actin-antibodies and were therefore of smooth-muscle origin [16]. Experiments were carried out on confluent monolayers of cells 4 days after pl ting in 12-well clusters (3.8 cm2). The cell density at the time of the experiment was 3.10 5 ce Is/well. fluxes has been described [17]. Permeabilized cells were exposed to anT~~~~8t~~k~f mzzk at 250s of the following composition: 100 mM KCl, 30 mM imidazole (pH 6.9)t+O.78 mM free Mg +, 5 mM ATP, 0.44 mM EGTA, 5 mM NaN3 and the indicated free Ca concentration. This medium was supplemented with 3 % polyethylene glycol in the experiments in which the effect of GTP was studied [3]. The transport process was stopped after 10 min by aspirating the radioactive medium and washin the monolayers twice with ice-cold efflux medium (100 mM KCl, 30 mM imidazole (pH 6.9s , 2 mM MgC12, 1 mM ATP, 1 mM EGTA and 5 mM NaN3). 1 ml of efflux medium, with or without 10 FM InsP3 or 5 PM A23187, was subsequently added to the monolayers and replaced elary 2 min over a 10 min period. The monolayers were then solubilized in 2 % SDS. The “Ca2+ present in each of the effluent samples and the remaining radioactivity in the cells were measured in a liquid scintillation counter.

Results The effect of adding investigated

10 /.LM InsP3 and of 5 ,uM A23187

in saponin-skinned

vascular

smooth-muscle

cells

to the efflux-medium after

loading

has been the cellular Ca 2+

pools for 10 min in a solution containing 0.14 PM free 45Ca2+ (Fig 1, closed symbols). 10 PM InsPg under these conditions released 88 % of the total of accumulated Ca2+, which was estimated by applying 5 PM A23187. This dose was maximal, since a second application of 10 PM InsPg or even of 50 PM InsP3 did not release any more Ca2+. Aortic smooth-muscle cells therefore contain at least 2 different Ca 2+ stores, which can be distinguished by their InsP3 sensitivity. The open symbols in Fig 1 represent the Ca2+ uptake from a solution containing 0.14 PM free Ca2+ and supplemented with 1 mM vanadate, an inhibitor of Y-type cationtransport ATPases 1181. Vanadate under these conditions inhibited the total Ca 2+ accumulation by 99.9 f 0.9 % (n=4)

(Fig 2a, closed symbols),

and therefore

inhibits

the Ca2+ accumulation

into both the InsP3-sensitive and the InsP3-insensitive compartments. Vanadate inhibited Ca2+ accumulation in the InsP3-sensitive Ca 2+ store with a KO.5 of 6 * 2 PM (n=5). 1184

the The

Vol.

174,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

hme

Fig 1. Effect monolayers

of

InsP3

and

A23187

RESEARCH

COMMUNICATIONS

(mm1

on

the

Ca2+

efflux

from

permeabilized

aortic

The cells were preincubated wit$5 (0 T+n symbols) or without (closed symbols) 1 mM vanadate for 15 min before starting the Ca uptake for 10 min in, respectively, the presence and absence of the inhibitor. or 5 PM A23187 were

The added

free [Ca2+] of the uptake medium was 0.14 to the efflux medium at time 0, as indicated.

inhibition of the Ca2+ uptake in the InsP3-insensitive 2 PM (n=5> vanadate. Fig 2b illustrates that 100 endoplasmic-reticulum

Ca2+ pump

by 97.0 f 4.1 % (n=4). uptake

from When

longer

a medium the [Ca2+]

completely

Ca2+ 2.5

uptake

Ca2+

(Fig could

of NaN3

from

was

not

2a, open

5 to 10 mM,

at 0.14 PM [Ca2+]

[19] inhibited

to 14 /*M, vanadate

symbols), was present

2b, open symbols) in

leaving

the Ca2+

did not any 16.3 f

4.9 % (n=4) thapsigargin,

accumulated

the mitochondria.

supplementing

still

20.1 f

and in 5 PM

proceed. Ca2+ which

sequestered

at 25 f of the

by 97.2 f 0.8 % (n=4).

was increased

(Fig

still

thapsigargin

InsP3

maximal inhibitor

Ca2+ sequestration

inhibitor

If 100 PM tBuBHQ

unaffected

inhibitors,

medium

uptake

of the Ca2+ uptake

of these ATPase concentration

0.14 PM [Ca2+]

Ca2+ uptake.

remained

% (n=4)

containing

of the uptake

10 PM

Ca2+ store was half PM tBuBHQ, another

the total

5 PM of the Ca2+ -pump

prevent

(n=5> of the control

[6], inhibited

FM.

Indeed,

the assay medium

5.1 % of the 25.2 *

in the presence increasing with

the

10 /*M

oligomycin plus 10 pM antimycin, or addition of 10 PM ruthenium red, did not affect this Ca2+ uptake (data not shown). This Ca2+ -compartment was InsP3-insensitive and its uptake process could not be inhibited by vanadate, thapsigargin or tBuBHQ. However, supplementing (al

Vanadate

ilog

Ml

inhibitors at two different [Ca2+] Fig Inhibition of the Ca2+ uptake by Ca2+ -pump After skinning, the cells were preincubated for 15 min with vanadate (a) or tBuBHQ (b) at a concentration indicated on the abscissa, followed by a 10 min loadi in an uptake medium or without the containing 0.14 PM (closed symbols) or 14 pM (open symbols) [y+ ‘$1 with inhibitor. The ordinate represents the inhibition of the total Ca uptake (mean f S.E.M. (n=4)).

1185

Vol.

174,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

lamb

Ka”l [Ml FM [Ca2+] dependency of the uptake into the InsP3-sensitive pools Results are expressed as means f S.E.M. (n=5). the

uptake

medium

with

ATP, completely inhibited inhibitors. At high free dependent

way

into

external free InsPj-sensitive PM

[Ca2+].

maximally

(10 U/ml) plus glucose (22 mM) in order to deplete the Ca2+ uptake that occurred in the presence of these Ca’+-pump [Ca2+], part of the Ca2+ was therefore accumulated in an ATPnon-mitochondrial

by the InsP3-sensitive

Ca2+ pool without

and -insensitive

involving

Ca 2+ pools as a function

[Ca2+] is represented in Fig 3. Both curves present a bell-shape Ca2+ pool has the highest Ca2+ affinity and is already maximally The

InsP3-insensitive

filled

Ca2+

hexokinase

an InsP3-insensitive

type Ca2+-pump. The Ca2+ uptake

and -insensitive

Ca2+

store

requires

higher

free

[Ca2+lo

a ‘P’of the

pattern. The filled at 0.5

and

became only

at 12 PM free [Ca2+].

Because the loading

of both

pools was impaired

at free [Ca2+]

exceeding

1OOpM (Fig 31,

Ca2+ release could underly this phenomenon. we did consider the possibility that Ca 2+-induced Elevating the free [Ca2+] in the efflux medium to 10 PM did not release any Ca2+, not even solution. Neither did 25 mM caffeine, an after 10 min incubation with this high-Ca2+ Ca2+ -release channel [20], release Ca2+. Because we routinely activator of the Ca 2i-induced used a [Mg2+] of 2 mM in the efflux medium, it is possible that this Mg 2+ would block the Ca2+-induced

Ca2+

release [20].

We have therefore

investigated

the effect of caffeine

in the

absence of added Mg2+, but also under these conditions no Ca2+-induced Ca2+ release could be demonstrated. Subcultured porcine aortic smooth-muscle cells, in contrast to primary cultures of Ca2+-release channel rat aorta [21], do therefore not express a Ca 2+-induced The different

Ca2+

uptake

sensitivity

into

to oxalate.

the

InsP3-sensitive

10 mM

oxalate

and enhanced

-insensitive

Ca2+ ’ pools

the Ca2+ pumping

into

presented

a

the InsP3Ca2+ store

sensitive pool by a factor of 4.83 f 0.35 (n=3), and that of the InsP3-insensitive by only 1.91 f 0.28 (n-3). Also DDTlMF-2 cells have a more pronounced oxalate-sensitivity in their InsP3-sensitive pools [3]. Both pools could finally be distinguished by their sensitivity to GTP. GTP promotes a 2+ rapid and substantial release of Ca through a mechanism different from that activated by InsP3 [22-261. The Ca2+ uptake of the InsP3-sensitive Ca 2+ store decreased by 52 * 5 % (n=5) 1186

Vol.

after

174,

No.

adding

InsP3

BIOCHEMICAL

3. 1991

50 PM GTP. The

and GTP therefore

observed

in NIE-115

AND

InsP3-insensitive

release

Ca2+ from

cells [25], but unlike

BIOPHYSICAL

Ca 2’

RESEARCH

pool was not affected

the same intracellular

parotid

COMMUNICATIONS

by 50 pM

Ca2+-sequestering

GTP.

pool, as

cells [27].

Discussion The

most

function pool

important

of the free

has a higher

pattern

of total

conclusion

[Ca2+]

this

work

is that

the

Ca2+

accumulation

presents

as a Ca 2+

a bell-shape pattern, and that the InsP3sensitive Ca2+ than the InsP3-insensitive Ca 2+ pool. Such a bell-shape accumulation has already been reported for rat pancreatic acinar cells

affinity Ca2+

from

for

[lo], in which neither evidence for Ca ‘+-induced Ca2’ release was found [28]. In these pancreatic cells the gating of the endoplasmic-reticulum Ca2+ -channels was unaffected by the intraluminal or cytoplasmic [Ca2+] [29]. The

finding

that

the InsPg-sensitive

[Ca2+].

Such data have already

Ca2+

pool is preferentially loaded at low [Ca2+], while the InsP3-insensitive compartment needs higher [Ca2+], implies that the fraction of Ca 2, released by JnsP3 should depend on the [Ca2+] at which the store was loaded, being lower at the higher directly data

to the uptake are compatible

14], they

with

can equally

pool at higher The nature

medium well

containing

been reported

for studies

various

of free [Ca2+]

levels

in which

InsP3 was added

[30-341. Although

these

a Ca2+ -dependent inhibition of the InsP3-induced Ca2+ release [12be explained by an impaired loading of the InsP3-sensitive Ca2+

[Ca2+]. of the non-mitochondrial

compartment

that

actively

accumulates

Ca2+ in the

presence excluded

of maximal effective concentrations of Ca2+ -pump inhibitors is unknown. We have the possibility that high intraluminal [Ca2+] would interfere with the vanadateinhibition [35], because two unrelated Ca 2+ -pump inhibitors left the same fraction unaffected, and because the same partial inhibition occurred after decreasing the free intraluminal [Ca2+] with

the

precipitating

anions

oxalate

and pyrophosphate

(unpublished).

Thevenod

et al. [lo]

observed that 16 % of the Ca2+ uptake could not be blocked by 2 mM vanadate in rat pancreatic microsomes, and presented evidence for a Ca 2+/H+ exchange-dependent Ca2+ uptake. It

is possible

vanadate-resistant specific) inhibitor proton-gradient

that

a similar

phenomenon

occurs

in aortic

smooth-muscle

cells,

because our

Ca2+ uptake was prevented by 0.5 mM N-ethylmaleimide, an (not very of the H+-pump supposed to be responsible for setting up the transmembrane [lo].

Acknowledgment L. M. is Senior

Research Assistant

of the National

Fund

for Scientific

Research, Belgium.

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