The separation of cell membrane calcium transport from extracellular calcium exchange in vascular smooth muscle

BIOCHEMICAL

Vol. 39, No. 4, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE SEPARATION OF CELL MEMBRANE CALCIUM TRANSPORT FROM EXTRACELLULAR CALCIUM EXCHANGE IN VASCULAR SMOOTH MUSCLE.

C. van Breemen and E. McNaughton of Clinical Pharmacology, Department of Medicine of Miami School of Medicine, Miami, Florida, USA

Division University

Received

March

16, 1970

The soundest should

method

be direct

with

relaxation

the function

problem

is

proach following

change

details

with

contractions

and before from

senses

of the

The principle

binding

sites

Lettvin charge

density

which

binds

(Takata

et al.,

pid

artificial

port

across

flux (1964)

have greater calcium. 1966;

This

this

latter

that

after

cells

calcium

with

the hope

to arteries.

This

of a reliable

method

calcium

apfor

extracellular

the cellular

while

into

membrane

the cells

predicted affinity

1969)

(van Breemen membrane

over

tissue

bound

calcium

fluxes

ex-

associated

for

sites

ions

for

period.

group

and van Breemen,

1969). it

time

the

any anionic

on the lobster

pH range

give

loss

higher

groups

567

then

the

due to their

and the negative

a wide

blocks

will

ions

up 45Ca,

out and displaced

the experimental

lanthanum calcium

has taken

lanthanum

radioactivity during

that than

was verified

the

45Ca is washed

The measured

Hafemann,

membrane

the ionic

considerable

isolates small

the extracellular

the cells.

et al.

which

relatively

is

by lanthanum,

calcium

with

to this

smooth muscle.

all

of 45Ca from within unidirectional

the

of the method

counting,

in tissues

interfering

approach

smooth muscle specific

patients

than

regulates

by the absence

a new method

and consequently

which

at sites

movements

We have designed

rather

An appealing

of the arterial

in the past

calcium

in hypertensive

smooth muscle, agents.

control

frustrated

cellular

pressure

of the mechanism

pharmacological

has been

spaces.

of arterial

in the myoplasm

of eventual

blood

of neuro-transmitter

to learn

concentration

of reducing

axon membrane of a phospholiBy studying

was shown

that

trans an

BIOCHEMICAL

Vol. 39, No. 4, 1970

optimum

affinity

transport

rate

cium should

the

squid

calcium

indicated 1969),

blockade

intestinal

van Breemen,

smooth 1970).

calcium.

across

As expected

in vascular

In this

num can be used to remove

report

at which

the

than

cal-

affinity

same time make the

lanthanum

completely

phospholipid membrane. Lanthanum membranes

and De Weer,

1970)

smooth

(Weiss

existed

much greater

mitochondrial

muscle

ions

and at the

the artificial

across

(van Breemen

a similar

calcium

transported,

for

fluxes

axolenuna

for

An ion with

poorly

unavailable

45Ca flux

blocked

groups

a maximum.

be very

sites

blocked

the

reached

thus

transport

also

of the negative

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Mela,

and strong

muscle

1968),

indirect

membranes

and Goodman,

1969)

evidence

(van Breemen,

and heart

(Palmer

we show how the above properties

the obscuring

effect

and

of extracellular

of lantha-

calcium

exchange.

METHODS Rabbits abdominal

were

aortae

solution

TS (in

killed

by a blow

rapidly mM/l:

removed NaCl 160,

5, pH 7.4,

bubbled

with

of all

and cut

transversely

fat

or 3 mm wide

strips

for

45Ca uptake: Na),

or K-TS

After

varying

periods

was used

adsorption ml Bray's

Sucrose-14C minutes

incubation

removing until

from

radioisotope

for

were

placed These

NA).

strips

were

in 1 mM LaC13 in

for

total

uptake:

moisture

extracted

KC1 4.6,

rings

for

dextrose were

10, Tris

then

contraction

in TS, Li-TS solutions

removed,

Ca determinations

with

cleaned studies

fluid,

was present.

by elution

after

with

blotted

in 2 ml of distilled

a portion 303

atomic in 10

counter.

and 37'

30 and 60 C.

on filter

was recorded water

45Ca.

and ashed.

5, 7, 15,

at pH 7.4, were

for

ash was dissolved

The wet weight

568

Elmer

scintillation

was followed

the strips

labelled

weighed

a Perkin

liquid

TS or KTS solution

were

substituted

For some strips

1 ml of the dissolved in a Packard

(Li

blotted,

N HCl.

The uptake

in either

physiological

The aortae

.l

and counted

the incubating

no residual

4 mm wide

and anterior

data.

spectrophotometer. solution

37O C).

into

thoracic

in a Tris-buffered

MgC12 1, CaC12 1.5,

strips

(K substituted time

and placed

temperature

flux

Aortic

The ash was dissolved of this

02,

on the neck and their

and

overnight

After paper and the in a

BIOCHEMICAL

Vol. 39, No. 4, 1970

shaker.

10 ml of Bray's 45Ca cellular

in Ca free blotted,

influx:

added

exposure

to 45Ca,

2 mM LaClj(La-TS)

weighed,

ashed

uptake

studies

in specific

was then

After

TS containing

All changes

solution

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

for

counting, aortic

one hour.

strips

were

The strips

placed

were

then

and counted. were

activity

preceded

by 10 minutes

due to mixing

with

Ca free

unlabelled

TS to minimize free

extracellular

calcium. Ca efflux: for

Aortic

20 minutes

mM LaC13. pared

for

before

They were counting

changed

were

allowed

to take

them in either

removed

up 45Ca from

Ca free

at varying

labelled

TS or Ca free

times,

blotted,

TS

TS with

weighed

2

and pre-

as above. Aortic

hooks

transducers.

placing then

Contractions: The lower

strips

were

connected

Transducer

by moving

rings

were

to aerators

and aerator

the mounted

mounted

ring

between

and the

were

rigidly

between

stainless

steel

hooks.

top ones to Grass

tension

connected

different

and solutions

were

beakers.

RESULTS Figure tic

strips

1 shows exposed

the total

to labelled

tissue TS over

calcium

and 45Ca labelled

a 100 minute

time

interval.

calcium

The exchange

Plabelled

L 20

40

60

80

100

minutes

Figure 1. of strips minutes. standard

tissue Ca concentration The uptake of 45 Ca and the total of rabbit aorta exposed to 4SCa labelled TS for hundred The distance between horizontal bars equals twice the error of at least four observations. 569

of aor-

Vol. 39, No. 4,197O

is rapid

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and appears

to plateau

20% of the

total

tial

45Ca exchange,

large

in TS, Li-TS

within

Ca inexchanged.

and K-TS

the

A possible

A comparison

(figure

first

2) with

20

lo-20

slow

of the

minutes,

uptake

total

tissue

the contractile

46 minutes

is

states

60

leaving

about

obscured

by the

labelled

Ca uptakes

of the aortic

ini-

rings

80

Figure 2. 45Ca influx in strips of rabbit aorta from physiological The distance solutions containing 160 mM Na +, 160 mM K+ or 160 mM Lii. between bars equals twice the standard error of at least four observations. Bars are absent if the S.E. is less than the radius of the circles. in

the same solutions

tween

these

the initial

(figure

two parameters. rapid,

large

3) demonstrates This

is

45Ca exchange

only

the total

of correlation

one of the reasons

occurs

5

lack

10

in the

extracellular

for

believing space.

15

minutes

Figure either

3. Tension 160 mM Li+

developed by rabbit aortic rings when exposed to or 160 mM ti containing physiological solutions. 570

bethat Other

BIOCHEMICAL

Vol. 39, No. 4, 1970

reasons

are:

1. There

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are many negative

fixed

such as on mucopolysaccharides,

proteins,

phospholipids

which

initial

of cell

45 Ca uptake

(Hudgins

following

solution continually

if

La ions was done.

recorded

calcium

rabbit

displace

Aortic

ions.

1968)

space and the

2. The rapidity uptake

tendon

for

of the

of sucrose-14C

shows similar

45Ca ex-

extracellular

Ca, the

coefficient

la2 could -z3 The concentration

(1969).

4

exposed

to either

1 mM 4SCa labelled

at 7.3. the total

bound

were

20 minutes.

and maintained space

this

strips

10 mN glucose,

in the extracellular

of Sparrow

bind

(Borle,

to the extracellular

would

1 mM LaC13 added

and the specificity

mucoproteins

3. Acellular

290 mM sucrose, with

in the extracellular

data).

experiment

containing

comparable 969).

(unpublished To determine

thod

is

and Weiss,

change

tions

surfaces,

sites

CaC120r

The pH of both Since

number

be determined of binding

was

the only

binding

according sites

the same

solutions

Ca and La were of negative

a solution

was 8.15

sites to the me0.3 mM/kg

1

20

40

60

80

r2mMLa 100

minutes

Figure 4. The loss of labelled exposed to 45Ca for 20 minutes, with 2 mM LaC13. The distance twice the standard error of the

Ca from aortic strips which had been to either Ca free TS or Ca free TS between the horizontal bars equals mean of at least four observations. 571

ca-

Vol. 39, No. 4,197O

wet wt.

BIOCHEMICAL

and the selectivity

of figure

4 also

That

displacement

this

the observation

rapid induced

extracellular TS before cellular

analyzing

and the outer

given

of between

the 45Ca which

in figure 0.06

The nature

These

aorta

rapid

is

La ions

only

membranes,

30-40

for

the cells. conditions

curves

saturable

stain

40 minutes

60

minutes

the

no more

of the

60 minutes

in LaCa

and Li and K substiof a rapid

which

phase

depends

is not

saturable linearly

entirely

80

Figure 5. The cellular uptake of 45Ca labelled Ca from physiological solutions containing 160 mM Na+, 160 mM Li+ or 160 mM F.?, measured after extracellular 45Ca was removed with La *. The distance between the horizontal bars equals twice the standard error of the mean of at least five observations.

572

the

and from

I

20

from

The labelled

consist

and uptake

Ca by La.

the same as the La

After

strips

curves

concluded

of the completeness

had entered

uptake

and 0.1 mM Ca/kg of the initial

tendon.

control

bound

is virtually

the aortic

under

5.

which

cell

To be sure

we left

The efflux

space dense

of the

rabbit

to the La-TS.

determined

extracellular

surfaces

45Ca loss,

Ca was 3.1.

of extracellular

the electron

the acellular

for thus

in the

that

45Ca displacement

are

on time.

place

(1970)

to be lost

influxes

tutions

takes

from

of La over

the displacement

of the La induced

45Ca loss

45Ca appears

phase

demonstrate

space

course

coefficient

by Smith

extracellular

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

clear

Vol. 39, No. 4, 1970

at the moment.

It

small

blood

after

the initial

present

which

entered

locked

in the lumens

or some calcium

calcium

solution.

the rates

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

calcium

by lanthanum 10 minute

calcium

with

may represent

vessels

in the control well

BIOCHEMICAL

free

exposure.

of tension

Ca uptake

in TS,Li-TS

and

the membranes does not

no contraction

of the linear

development

penetrated

In any case it

since

the cytoplasm,

The rates

which

of capillaries

re-

was illicited

correspond

very

and K-TS shown

in figure

DISCUSSION The exigency trated

by two simple

(Murphy chi

of our

et al.

(1969)

plete

would

measurments traction

mM/l

be they

and that

tissue

uptake

led

used

to false

described

Ca influx

cells

that

during

the high

for

K con-

water

to 275 was 1.5

4.2 mM/Kg.

Thus

was 3.0 mM/Kg,

that

* 1.5 mM/Kg = 3.45

alone

resulted for

com-

calcium

from 440 ml/kg

concentration

. 4.2 mM/Kg + 0.275

by Bian-

such small

tissue

solids

required

given

7uM Ca/kg

of the extracellular

that

artery

in total

and extracellular

than

values

obvious

decreased

of the contraction

grater

is

Secondly

calcium

was 0.725

in the

solutions.

by 4 UM Ca/kg development would

past

correlations in this

in quantitive

K and Li

figure

It

by the variation

concentration

may be illus-

full

mM/Kg.

in an arteffectual activation

of the con-

proteins.

method

tension

mechanism.

labelled

effect

64 times

Methods

trol

aorta

the mechanical

tractile

have

calcium

coupling

no more than

space of our preparation

the relaxed

calcium

require

exchange

of 5mg actomyosinfg

for

or unlabelled.

of the remaining

of the contracted Thus

would

calcium

a value

required

obscured

labelled

The labelled

whereas

the aorta

be completely

cellular

Using

of the contractile

the sucrose

ml/kg.

for

and the calcium that

activation

amount

calculations.

1969)

we find

new method

between

paper agreement

Potassium * minute during

ignoring

calcium

eliminates with increased

and lithium high

one or both

slope

development

of 45 Ca uptake

over

* minute.

as calculated

65% of maximum/minute

573

The

and gives

of tension

by 0.8 uM Ca/Kg

considerations

and contraction.

difficulties

the rates

K depolarization

be 57% of maximum/minute.

fluxes

these

the

of the above

rates

in high the

The rate from

of

conof

the above

was observed

in

3

BIOCHEMICAL

Vol. 39, No. 4,197O

figure

3 during

and observed

the fast

rates

phase

of contraction

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the contraction. in the Li

substituted

The respective solutions

calculated are

11% and

10% of maximum/minute.

This investigation National Institute Association.

was supported by PI-IS Research Grant No. 5688 from of Health and Grant No. 69AG30 from Florida Heart

REFERENCES

Bianchi, C.P., Fed. Proc. 28, 1624 (1969) Borle, A.B., J. Cell Biol. 36, 567 (1968) Rafemann, D.R., Comp. Biochem. Physiol. 29, 1149 (1969) Hudgins, P.M. and Weiss, G.B., J. Physiol. 217, 1310 (1969) Lettvin, J.Y., Pickard, W.F., McColloch, W.S. and Pitts, W., Nature 202 1338 (1964) Mela, L., Arch. Biochem. Biophys. 123, 286 (1968) Am. J. Physiol. 217, 666 (1969) Murphy, R.A., Bohr, D.F. and Newman, D.L., Palmer, R.F. and van Breemen, C., Clin. Res. 18, 1, 26 (1970) Smith, D.S., Personal communication Sparrow, M.P., J. Physiol. 205, 19 (1969) Takata, M., Pickard, W.F., Lettvin, J.Y. and Moore, J.W., J. Gen. Physiol. 50 461 (1966) van Breemen, C., Arch. Int. Physiol. Biochim. 77, 710 (1969) van Breemen, C. and De Weer, P., Nature in Press van Breemen, D. and van Breemen, C., Nature 223, 898 (1969) Weiss, G.B. and Goodman, F.R., J. Pharmacol. Exp. Ther. 169, 46 (1969)

574