Effects of A23187 and Ca2+ on volume- and thiol-stimulated, ouabain-resistant K+Cl− fluxes in low K+ sheep erythrocytes

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 14, 1984

Pages 790-796

EFFECTS OF A23187 ODABAIN-RESISTANT

AND K+CI-

Peter Department

Received

of

November

2,

K.

Ce 2+ ON VOLDMEPLDKES IN LOW Leaf

Physiology, Durham,

end

A.

AND

K+

Yengor-Jensen

Duke University Carolina

North

TRIOL-STIMDLATRD, SRPEP RRYTRROCYTRS

Medical

Center

27110

1984

Onebein-resistant (OR), Cl--dependent K+ (K+Cl-1 transport measured by Rb+ influx in isosmotic end anisosmotic media wes stimulated by the Ca2+ ionophore A23187 and EGTA (ethylene-glycol-tetracetic acid) in low K+ (LK) but not in high K+ (EK) sheep red cells. Increasing external Ca2+ concentrations, 1ce2+1o, from about 10s7 to 10m3M in presence of A23187 end in absence of ETA inhibited OR Rb+ influx, in LK red cells osmotically shrunken or swollen es reagent N-ethylmeleimide (NW). Hence the well es treated with the thiol vo lume and the =,-stimulated K+Cltransport system in LK cells can be interact experimentally modulated by cellular Ca2+ or other Me2+, which may with on the K+Cltransporter under the control of membrane sulfhydryl sites 0 1984 Academic Press, Inc. (SE) groups.

Treatment cally

with

LK but

presence

of

volume-

end

and tem

on

of

Cl-,

immunological

effects, that

treatment

K+Cl-

transport were

red

of

we

in

inhibitory contrasts

This work was presented Sympos ium on Volume Switzerland, September Red Cell Club Meeting

of

LK

red

control of

with

fluxes

whether

cells not

the in

volumeother

in pert Regulation 4-6. 1984, in Montreal,

0006-291X/84 $1.50 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.

by

out of

reports

same

We

cells,

effects

and while

thiol-dependent

K+Clof

Both,

transport

is

these

the

electroneutrel,

LK

finding

the International in Crans Sur Sierre, end in pert at the October 20, 1984

790

be

the

A23187

on

(11).

unmodified

levels.

geneti-

dependent

reversibly

ionophore

that

et

the

the

NRM-treated end

flux

to

by

this

of

furosemide

considered

Ca2+

with

swelling

e OR K+

abolishes

intracellular

but both

are carried

investigated

changes of

fluxes

cells

osmotic

inhibited

OR K+ClLK

or

activates end

probably

depletion

es

Na+.

K+Cl-

basal

NRM

cells

of

the

(121, such

reagent

grounds, in

flux

observation

RK sheep

NRM-stimulated

metabolic

K+Cl-

SE-group

independent

involved

Since

Ca2+

not

the

syscell

(13).

NPM-activated based now

on

other

demonstrate

EGTA

stimulated

A23187 transport. agents

plus Our were

Vol. 125, No. 2, 1984

nonspecific

in

BIOCHEMICAL

these

on volume-dependent tumor

cells

K+

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cells

(3,5),

and

with

fluxes

in.

amphiuma

mostly red

activating

cells

(21,

effects

of

lymphocytes

Ca2+

(8).

and

(91.

MATERIALS

AND

MFTBODS

Dorset sheep served as donors of genetically homozygoos. and immanol og itally LL type LK red cells, respectively, with cellular K+ concentrations, ranging from about 14-25mWL.cell water. For all experiments cells were [K+l,, washed in an isosmotic (-295 q OsY) solution of the following composition (mR): 155 NaN03, 10 TrisfMOPS (tris-hydroxymethyl-aminomethane/2-N-morpholinopropane

snl

washing

fonic

and

acid)

preincubating

+

0.1

in

NO;

oaabain,

pH

media

was

7.4

to

at

O’C.

The

K+

leaks

keep

purpose

of

through

cell Cl--

For incubation with 1mM NEM, an alidependent transport at a minimum (101. quot of packed red cells was subsequently removed and treated as described elsewhere (10,141. Al iquot s of 2 ml packed. untreated or NEWtreated red cells were added to preincubation flasks containing 18ml of the above solution with the following additions: 1. none; 2. 50~1 of 0.2mR-0.W Ca2+ acetate; 3. 60 or 100~1 of 1.9mM A23187 (free acid, Calbiochem. Corp.); 4. 100~1 0.2R EGTA pH 7.6; 5. 2 and 3 combined; and 6. 3 and 4 combined. The final concentrat ions of Ca2+, A23187 and EGTA were (M) : 10-6-10-3, 6-9.5x1O-6. and 10b3, respectively. After incubation divided into 2 centrifuge tubes and the supernatants removed Cl--dependent and -independent were mixed at t=O with Clflux flux medium (-300 mOdI). to NaCl (A)

for

15

minutes pelleted

(A,B),

at 37’C, cell suspensions were by centrifugation at 12,OOOxg. To measure Rbf influx through the pellets in test tubes labelled A and pellets in test tubes B with NO?j

completely. K+ pathways. medium A,

B, both equilibrated hyposmotic (-240 or NaN03 (B) while

at 37’C. mOsY), and concentrations

Flux media hyperosmotic of the

A and B were i sosmot ic f-370 mGsY) with respect Tris/RGPS buffer (1omM,

pH 7.4 at 37’C) and Rb+ (1OmM) were kept constant. In addition, tubes A, B of [Ca2+l flasks 2 and 5, and of flasks 4 and 6 contained the same and of [EGTAI , respectively, used during the preincubafion step, to e&re their presence throughout the flux period. One ml triplicates were removed at 1 hour after t=o. washed in ice cold isosmotic MgC12 solution, Tris-MOPSbuffered at pH 7.5, and hemolyzed for determinations of Rb+ uptake. Calculations

of

ouabain-resistant

The Cl--dependent i OR measured %b

Since

effect 1

in

of

medium

the

swelling

Cons in

is

cells

lack

dog

red

presence 9.5gM of istent

240mGsM

were

referred

described to

as

elswhere

the

(10).

difference

of

respectively.

AND

fluxes

media. or

in

ic,

RESULTS

and

of Rb+

B,

red

Ca2+

influx,

(‘~~lAcl

A and

(7)

effects on

anisosmotic

human

external the

lmMICa2+lo

influx,

sheep

for

show

370mOsM

Rb+

adult

described

Rb+

DISCUSSION the

cells of

with

(171,

A23187

A23187 LK

Ca2+

in red

absence cells

caused

791

it

was

or

feasible

the

channel

(1)

to

the

Data

presence

of

measured reports

K+

fluxes.

on OR K+

earlier

solutions

induced

in (3,s)

cell

Cl--dependent

study in

Table

1mM EGTA

isosmotic

or and

shrinking Rb+

in flux

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE

Effect of A23187, EGTA and Ca++ on Ousbain-Resistant Osmotically Shrunken, Normal, and Swollen LK Sheep

The

Influx

in

370 Pretreatment

Anion

Control

0.23

300

m0s.U

(ihf$)Acl

* SE

Cl

f

NO3

0.12

0.02

Cl

0.53

0.05

1OpM A2187* 1mM EGTA

0.16

Cl

0.55

Cl

0.09 0.95 0.04 0.11 0.84

Data

derived

from

4

LK 259 except for influx experiments.

of

component

caatly

when,

of

dependent

not ineffective

be

specifically

to

reduce

basal. well

in

Or

RK red

stimulatory ca2+,

K+Cl-

Rb+ lacking

end r8Sp8CtiV8iy.

1.6

and

normal

and

swollen

was

present

in

The

and loop

LK

not

OR K+C!l-

inhibitory

red

the

0.26

0.06

0.15

0.06

fold red

0.64

0.09

0.92

0.03

0.89

-

0.11

0.03

addition

to in

three

Ca++ since

also

prevented

shorn).

Furthermore,

and

increases

of

respecHowever.

A23187.

Ca2+

farosemide

A23187

1mM EGTA.

all

zero.

cells,

sigaifi-

cells,

the media

or

Cl-tested,

FiGTA

effects

to known

K+Clthe

alone

appear

anti-Ll,

ouabain-resistant

fluxes

enhancement

no

effects

were

transport.

effects oa

0.04

4.6

mediated

NH&stimulated

(data

0.24

9.5uM

of

from A23187

diuretic

flux

0.06

without

omission

inhibited

of

1.15

with

1.4,

the

transport

the

However,

with

shown).

0.03

respectively,

different

not

as

cells

media.

completely

volume-

Cl--dependent

The mTA

(data

es

NO5

significantly

on

(13,15,161

seen

being

were

2 Rb+

reduced

1mM Ca2+

fluxes

of

of

decrease,

shrunken.

slightly

EGTA,

Rb’

(YZXl

in

were

instead

or

in

cells

mean

preiacubatioa,

observed,

which

red

the

increase

transport

during were

tively,

to

K+

on

are

0.16

0.01 0.12

experiments

controls

present

#3*c1

of

0.13

numbers

0.07

* SE

0.19

0.02

0.01

where

l

affecting

EGTA

were

0.02

independent

the

0.80

0.72 0.13

0.10

OR

(lMRb)bC1

1.08

-

0.10

m0s.M

SE

0.84

0.03

0.20

NO3

f

0.46

0.09 1OpM A23187 1mM1ca++1

Cells 240

(ie)AC1

0.55

0.46

Rb+ Red

mOsM*

i$

0.02

0.36 NO3

SE

0.02 0.10

1OpM A23187 (-10-71 ca2+j

1

OR Rb+Cl-

792

of

A23187 flux

in is

clearly

combination illustrated

with in

Vol. 125, No. 2, 1984

Figure

1 where

affected

by In

in

the

free

can

presence of

concentrations

thus

at act

shows

as a

high

swollen

ing

[Ca 2+10

or

ETA)

the

i OR ldRb

1:

[Ca2+lo,

Ca2+ K+Cl-

i OR MRb as shrunken

may

after

(see

strongly

NO;,

6

The effect resistant

nominally

enter

from

influx

is

Ca2+-free cellular

maximally

of

the

inside

infinity

fluxes point

supporting

the

ionophore

membrane.

A23187. were

on

abscissa).

for

LK

of

sheep

that

for

1mM Ca2+ (closed cells where the

of in

symboIs). fluxes were

A23187

the

by

was

an

A23187

mediated

on

ouabain-

The solid measured

of

FGTA

and broin 370 and

respectively.

‘Ike effect of A23187 and varying [Ca2+lo on ouabain-resistant. Rb+ influx in osmotically shrunken and swollen LK sheep red i OR cells. OR Rb+ influx, MRb was measured in 370 (filled squares) and 250 m0s.M (open squares) medis after pretreatment in solutions presence 10-31.

cated.

with

and of Fluxes

Bars

without FGTA were = range

6pM A23187 (--point x-axis) measured either of 2 experiments.

793

(as in

indicated and [Ca2+Jo Clor NO3

on top). from 10m7

media

as

red

A23187

There

presence

2

increas-

activated

concept

and

Figure

Note

which

its Con-

9 or

media,

the

and

flux.

the

[Ca2+Jo 6pM

of various conce;t;fftions Rb+Clinflux, ( %blACl’

symbols) lines

of

(i.e.

altered

@Cl-

through

extracellular

Rb+

is

OR

cell

with

solutions

Ca2+ of

the

treatment

inhibited

240 m&M 2:

Rb+

activation

flux

media

in

in

of

function

A23187 3 [FM)

(open ken

Figure

Cl-dependent

distribution permitting

Ca2+-free

01 01

the

and

lowered

progressively

on

that

ionophore

of

of

nominally

Figure

seen

of

inhibitor

plot

cells

effect

be

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A23187.

presence

versely,

in

it 6nM

tbe

BIOCHEMICAL

indi-

in to

no

Vol. 125, No. 2, 1984

Ca2+

modulation

values

at

which

about

one

order

(10S3M) red

of

sport affected

free cell

and

cells higher

When

of of

to

apart

swollen fluxes

controls

the

Ca++

in

absence

than

Me2+

shrunken in

the

and

shrunken sug-

K+Cl-

the

site

on

Rb+

Ca2+

effect

in

isosmotic

the

was

Rb+ least

of

as

varying

not

[Ca2+lo

Note

that

to

the

inhibition

were

component if

and

compared

further

effects

one,

A23187 medium.

absence added

No

at

be

seen

was

modified

two

Ye2+

Rb+

Rb+Cl-

by

in

Ca2+.

binding

Rb+

of

and

flux

fluxes

NO;

These

sites

A23187

occurred

mainly

media, data

affected

thus suggest

by

the

media

as

NEM

f

Ln mV7

I 6

/

-LOG

The Rb+

effect

ofiA$i18’I

and

I 5

I 4

I----L--3

[Cd+],(M)

varying

IO NEWpretreated

func t :zr KY% o III absence ence of A23181 is indicated were determined in either Clof 2 experiments on 2 animals.

794

or on or

1ca++1 cl

on

cells

in

isosmotic

of

6pM A23187.

presence the top NOT media.

of

onabain-resistant

the Bars

of

NRR-stimulated

presence

of

on

the

flux

A23187.

3:

tranmay

Cl-

Figure

were

finding

between

between

A23187 and

This

relationship

equilibrium

of

required

activity.

[Ca2+lo

The

(10-4MI)

was

baseline the

only.

swollen

less

govern

the

route

between

to

laws that

the

cells

the

in

Cl--dependent

presence

equal

transport

volume.

[Ca++l,=>lO%I.

the

in

action

3 shows

was

Rb+

magnitude

Rb+

Me+

NRR-treated

ence

about

inhibit

Figure

RGTA .

OR was MRb

mass

by

fluxes

Cl--dependent

of

to

and

the

Hence

that

gests

at

i

cells.

cells

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

figure. indicate

PresFluxes range

only the pres-

Vol. 125, No. 2, 1984

One in

must

these

inhibitory

we

fully

in

sheep

data

may

and

red

cells

no

suggest

effects

A23187

and

verted

into

not

shown)

alter

the

which

reported

of

and

thus ratio

EGTA

were

was

found

both

cell

cellular

ATE

metabolic

intermedi-

reported

to

reduce

that

probably

explains

the

K+

cotransport

(6).

How-

Na+.

K+Cl-

fluxes

other

bivalent

or

supported were

inhibition

(41,

an observation

exposure

bound/free

of

in

the

Ca2+

which

reduce

membrane-bound

on

the

selectivity

A23187

into

of the

in

Ca2+

LK

the red

the

findings

that

subsequent Ca2+

cells

to

A23187

Ca2+

membrane

and

for

with con-

EGTA

(data

itself

may

SE

NBB,

ghosts Me2+

stimu-

was

NEM

including

site

mem-

the

A23187

effects.

cell

by

the

regulatory

red

as

carrier

membrane

reagents,

well

treatment and

alkylating

human

same with

by

the

as

cat ions

Ca2+-binding

SH group

changes

flux

by

membrane

several

our by

of

cellular

participate

by

K'CI-

of

volume through

abolished

reversibility

Indeed

Caz+

on

effect

Ca2+

and

indicating

flux.

some

a fact

that

strongly

upon

of

by

(4).

ionophore

such

lowered

through and

cells

its

is

that

stimulation

progress

introduced

A23187

Ca2+,

to

A23187

effectively

shown).

of

hypothesis

K+

or

ouabain-resistant

somehow

dependent

directly

possibility

interaction

This

latory

not

the

Ca2+

red

Ca++

(data

the

brane.

human

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

plus

Indeed,

of

trigger

modulating

hence,

-

levels

confirmed

Our NEW

‘%),l

effects for

A23187

and (’

ATE

ever,

that

experiments

cellular

in

consider

inhibited

ates,

BIOCHEMICAL

groups

for

Cl--

have

been

(18). other

Work than

is Ca2+

cell.

ACKNOWLEDGEMENTS The ting

of

supported the

Norske

excellent the by

technical manuscript

US PHS

Shell

grant

Oil

assistance by

Ms.

AM

28236

Gay

of Blackwell and

for

Mrs.

Kristen is

a small

A.

Huber

acknowledged. part

by

and

typeset-

This

work

grant

83-524

was from

Company.

BEFEBENCES 1. 2. 3.

Brown, A.M., J.C. Acta 511:163-175, Cala. P.M. J. Gen. Dunham, P.B., and

Ellory, J.D. Young, and V.L. Lew. 1978. Physiol. 82:671-784. 1983. J.C. Ellory. J. Physiol. 318:511-530, 795

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1980.

Biophys.

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6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

BIOCHEMICAL

AND BIOPHYSKAL

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