A chloride dependent K+ flux induced by N-ethylmaleimide in genetically low K+ sheep and goat erythrocytes

Vol. 92, No. 4, 1980 February

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

27, 1980

Pages

1422-1428

A CHLORIDE DEPENDENT K+ FLUX INDUCED BY N-ETHYLMALEIMIDE IN GENETICALLY LOW Ki SHEEP AND GOAT ERYTHROCYTES* P. K. Lauf Department Center,

Received

January

and B. E. Theg

of Physiology, Durham, N.C.

Duke University 27710, USA

Medical

3,198O Summary

In genetically low K+ but not in high Kt red cells of sheep and goat Nethylmaleimide induced a ouabain insensitive K+ flux as measured by tracer influx or net efflux methods. The augmented K+ flux was observed in Cl- or Br- but not in NOj, SO2-or PO2- media. The action of N-ethylmaleimide was distinct from that of par ? chlorofiercuribenzoate or its sulfonic acid derivative which increased both passive Kf and Na+ movements across the red cell membrane. The instantaneous selective action of N-ethylmaleimide suggests that sulfhydryl groups control a K+/Cltransport system which, associated with the low K gene, is apparently functionally silent in adult ruminant red cells. Ruminants

such as sheep,

K+ (HK) or low dominant

Kt (LK)

gene

tained

(3).

sheep the

content

passive

(4,

NatKt

devoted

Na+K+ leak

pathways.

cation

behaves

(9,

passive

permeabilities

in sheep

lo),

(4)

Although groups

cells,

very

is

to both red cells

N-ethylmaleimide

involved are

In LK

(8).

in these

parameters

has

of the

the properties

of the groups

transfer

cells

while

As there

of monovalent

known to indiscriminatorily

increase

(11)

as well

as

(12). (NEM) is widely

to affect

lil; 1980 b-v Academic Press, Inc. in an-v form reserved.

Na+K+ pumps,

and sulfhydryl-(SH)

in passive

Nat and K+ ion

used in studies

the Na+K+ ATPase

as poster at the annual Fall New Haven, Nov. 10, 1979.

of reproduction

are main-

in HK cells,

proportional

amino-

0006-291X/80/041422-07$01.00/0 Copyright AN rights

than

high

by a

magnitudes.

greater

known about

either

controlled

7) different

the biophysical

little

with

compositions

of distinct is

In human red cells

and has been shown

* Presented University,

fluxes

is

state (6,

inversely

and organomercurials

and goat

steady

to characterizing

have been shown to be functionally cations

have red cells

The LK property

K' permeability

Nat permeability

pumps in these

2).

Na+K+ leak

the passive

been much effort

and cattle

5) and quantitatively

insensitive

red cells

(1,

The different

by kinetically

and by ouabain

goats

1422

meeting

(13)

of membrane

and the Na+/Na*

of the Red Cell

Club,

SH or

Yale

Vol.

92, No.

counter

Na+/Li+

Nat ground

selectively HK red cells reside

the control

transport

(14),

in these increases of adult

AND

in sheep

permeability

Kt permeability

groups

BIOCHEMICAL

4, 1980

red cells

no studies

cells

(14,

on the

RESEARCH

Cl - ion

and goat

action

in a funcationally of the LK gene in

silent these

Material

K'/Cl-

altering

of NEM on the

Here we show that

dependent

suggesting

COMMUNICATIONS

15) without

have been reported.

an apparently sheep

BIOPHYSICAL

that transport

Kt flux

passive NEM

in LK but

NEM reactive system

the

not

membrane which

is

SHunder

cells.

and Methods

Blood was obtained from Dorset sheep and Nubian goats whose red cell K' and Nat ion concentrations had been determined previously by atomic absorption spectrophotometry (Perkin Elmer, Model 460). Chemicals were of analytic grade NEM and the organomercurials parachloromercuribenzoate (PCMB) and its sulfonic acid derivative (PCMBS) were purchased from Sigma Chemical Co., St. Louis, MO. and dissolved in the experimental solutions immediately prior to the start of the experiment. Efflux Experiments: Prior to each experiment, cells were washed in 280 mOsm Tris/Cl buffered [pH 7.!)+choline-Cl containing lo-4M ouabain to exclude the co$r;;u;;i!is.by the Na K +pump. When Cl- ions were replaced by BY, NO?, ions their Na salts were used and the pH adjusted to 7.4 with Tr?s base ritrated with the respective acid. At zero time packed cells were injected into incubation flasks with either of the above media -I 1 mM NEM, 0.2 mM PCMB or PCMBS, to give a final suspension hematocrit of about 5% (v/v). Incubation was carried out at 37OC in a shaker bath and samples were withdrawn at various time intervals, centrifuged for 1 min in a Sorval RC5B centrifuge, and the cell free supernatants were analyzed for hemoglobin (OD$*7) and monovalent cation concentrations, [Cat],. The [Cat], values were corrected for cation release due to spontaneous hemolysis (less than 1% hr-1) using the ratio of 0D527 to 00527, the theoretical optical density at 527 nm of 1 ml hemolyzed pscked red'gells. An aliquot of each suspension was hemolyzed with a detergent to provide the equilibr'um52~,0D5$q~entration c of,hemoglobin, (ODz27), (OD The equilibrium cation concenwas measured ig the !$me sample. For very low hematocrits the cation concentration in the extracellular Hence for Kt and Nit Release the ratios of [Klt/[Klt'" and [Nalt/ [Nalt'" were calculated from which the efflux rate consfants'(corrected For the ?ime interval required for removal of supernatants) were computed using the first order rate equation

-In

(1 _ [Cat1h

) = Okcat?.

[Cat]&=" Influx Experiments: Kt influx was measured with 42 [Kl as tracer (Burlington Research Facilities, Raleigh, N.C.) in Tris/Ci buffered NaCl media (280 mOsm, pH 7.4 at 37OC containing 5 mM K'/Cland lo- M ouabain -f 1 mM NEM. Experimental details of this technique have been published elsewhere (6, 7, 16). Influxes were computed in mnoles K+/L. cells x hr and converted into influx rate coefficients by dividing the ouabain insensitive Kt influx by the intracellular Kt concentration.

1423

Vol.

92, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

Table

RESEARCH

COMMUNICATIONS

1

EFFECT OF NEM ON OUABAIN INSENSITIVE 42K INFLUX RATE COEFFICIENTS IN LK AND HK SHEEP RED CELLS

Cells

[Kl

(n=3)

mM/L.

LK(LL)

16.5

+ 1.0

0.052

t 0.008

0.254

+ 0.066

4.88

HK(MM)

80.9

t 6.8

0.028

+ 0.005

0.035

t 0.006

1.25

and

Discussion

Values

ikK

C

Cells

2 SEM

We first rate

Table was

noticed

the

that

of in

ikK, NEM

statistically

corroborated

effect

coefficients,

1 shows no

Ratio: NEM Control

NEM (1 mM)

Control

Results

influx

(Hr-')

increased

K+ net

creased

the

K+ efflux

control

'kK

which

Figure

1 may

be due

is

constant,

similar

to

on

tracer

'kK

in

in

control

The

between

sheep

cell

HK cells.

larger as

that 8 fold

NEM well

while This

1 shows by

of

as

the

K+

specimens.

LK cells

LK cells

'kK.

analysis

red

in

Figure

'kK,

differences

the

5 fold

measurements.

rate

in

3 HK sheep

almost

effect

efflux

to

3 LK and 'kK

significant by

1 MM NEM

finding

was

2 mM NEM inabove

effect to

there

the shown

the

in

freshness

LK 71 0.5

-

30

45

60

75

90

MlllUWS

Figure 1: Effect of 2 mM N-ethylmaleimide on K+ efflux rate constants. LK sheep red cells were washed in isotonic choline chloride medium, Tris/Clbuffered to pH 7.4 and containing 10-4 M ouabain. At zero time packed cells a final suspension hemawere injected into the same medium ? 2 mM NEM to give Samples were withdrawn after given time intervals tocrit of about 5% (v/v). Calculation of data of incubation at 37OC and analyzed for released Kt ions. as given in Methods. points and of the OkK values

1424

Vol.

92,

No.

BIOCHEMICAL

4, 1980

AND

BIOPHYSICAL

Table EFFECT

RESEARCH

2

OF 1mM NEM ON Kt AND Nat EFFLUXES FROM 8 GOATS

Cells

[Klc

[Nalc

(n)

mM/L.

Cells

HK

75.7

13.5

NEM

0.022

IN RED CELLS

Ratio: NEM Control

OkK(Hr-l) Control

COMMUNICATIONS

OkNa(Hr-l) Control

0.033

Ratio: NEM Control

NEM

0.145

0.159

20.024

50.031

0.052

0.054

1.50 (5)

f 4.8

+ 1.4

28.5

65.9

LK

io.003

1.10

to.008

0.064

0.176 2.75

-r 1.4

(3) Values

of

the

NEM

lot

0.006

for and

ficant

ability of

red

support

which

appear

red

the to

prevent

the

surprising

LK

PCMB

nor

cell

in

kO.004

change

in that

LK sheep

and

from

in

light

As

expected

(14),

K+ and only. of

occurred

in

The their

with

functionally

effect

1425

shown).

Kt

perme-

both

alter-

LK and of

NEM.

membrane

HK goat The

data

SH groups

with

part

usefulness

be

buried

within

of

the

organomercurials.

shown),

known

+

signi-

significant

presence

NEM may

effluxes

dual

in

some

no

the no

0.036

was not

associated

binding

not

Nat

OkKvalues

the

cells.

with

(data

(data

was

'kNa

SH groups

NEM

there Also,

red

there

selectively

5 goats.

reacts

the

since

1 mM NEM were

cells

from

reacting

of

increased OkK

of

red

used

comparison,

presence

while

goat

those

In

increased

NEM and

concentrations

and

HK sheep

cells

SH groups

affected

1 mM NEM.

specimens

HK red

action

NEM

1 mM NEM also

hypothesis

in

different

NEM which

absence

be genetically

putative

and

in

significant

and

PCMBS

not

3 LK goat

permeability The

not

to.006

different at

in

in

the

respectively.

that

no

shown

'kNa

efflux

to

cells

2 reveals

Kf

cells

brane

1.04

iO.027

achieved

+ 0.005, on

in

not

were

0.036

effect

ation

and

3 HK red

Table

Kt

used

effects

SEM)

ko.009

f SEM

saturation (A

+ 1.7

and

as

shown

indiscriminately of

the

two for

PCMBS in

Table as

opposed

organomercurials cation

mem-

exchange

3,

did both to is

experi-

Vol.

92, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

Table

RESEARCH

COMMUNICATIONS

3

EFFECT OF NEM AND ORGANOMERCURIALS ON K+ AND Na+ EFFLUXES IN LK SHEEP RED CELLS

Treatment

Choline

'kK(Hr-')

Cl

1 ti

NEM

Ratio

'kNa(Hr-')

Ratio

0.058

+ 0.006

--

0.037

2 0.005

--

0.227

f 0.031

3.91

0.042

+ 0.005

1.14

0.2

nn'! PCMB

0.121

+ 0.016

2.09

0.086

t 0.009

2.32

0.2

mM PCMBS

0.156

f 0.026

2.70

0.174

t 0.014

4.70

(n=4)

+ SEM Table EFFECT OF NEM ON OUABAIN OF LK SHEEP RED CELLS IN Cl-

4

INSENSITIVE K+ EFFLUX SUBSTITUTED

Nat

MEDIA

‘kK (Hr-l) Anion

Ratio:

Control

NEM

NEM/Control

f

cl-

0.073

+ 0.012

0.270

+ 0.047

3.70

*

Br-

0.073

+ 0.007

0.303

+ 0.072

4.15

j-

NO3

0.061

+ 0.020

0.068

* 0.017

1.11

*

so;-

0.081

+ 0.010

0.100

+ 0.014

1.23

*

PO;-

0.067

+ 0.019

0.083

+ 0.013

1.24

n ? SEM t n=6,

* n=4,

f SEM

ments

(4,

11,

12).

groups

at

the

surface

Since than

that

efflux that

of

the

Kt

not

affect

their later

permeability

of

(17),

we were

the

presence

of

Cl-

on or

the

OkK

Brouabain

ions

proposed

within

ions

NEM effect for

and

passive

required

stituted did

the

Kinetically,

was in

the

membrane

Cl-

ions

surprised

external not the

insensitive

mode

Cl-

observed incubation ground

1426

attack

is

first

on

SH

(9).

in

to

of

red

find

cells that

the

(or

Br-)

ions.

when

NOj,

SO:-

media. permeability

is

This

much NEM

Table and

PO4*-

anion for

greater

augmented 4 shows ions

sub-

substitution Kt

K+

ions

as

Vol.

92, No.

shown in the is

BIOCHEMICAL

4. 1980

controls

independent

of the

of the fast

AND

table.

BIOPHYSICAL

The NEM induced

Cl-/Cl-

self

exchange

4-acetamido-4'-isothiocyano-stilbene-2, duce

the augmented

in human (18) about

25-30%

(data

shown to be present Ehrlich

Ascites

studies

are

Kt and Cl-

control

in these

transport

sheep

and goat

tion

of its

type

reticulocytes

peripheral

It

One is

Cl-

possible

tempted

perhaps

are

cell

findings

is possible

that

old,

down-regulates

the

may play its

Kt

K+/Cl-

of the

which

state

for detailed of

presence is

an adduct which

under with

the an SH

may also

silent

be

in adult

LK cells

are

flux

as they

enter

immature

role

the ques-

adult

LK cells

transport

an important

has been

the LK gene raises

Kt leak

in their

steady

ions

by

(23).

in the adult

absent that

cell higher

Perhaps

Kt efflux

of transport

functionally

with

not re-

Although

system

cells

system

have a tenfold

functionally

red cells

red

mM

cotransport

the vestigeal

NEM forms

association

did

Nat/K+

(22).

Kt transport

LK sheep

0.1

has been proposed

suggest

and goat

(24).

(SITS)

and stoichiometry

The precursor

to speculate

evolutionary

our

and its

which

which

linkage

since

Kt and Cl-

and also

the

transport

role.

acid

for

(20)

dependent

(17)

Kt efflux

the NEM effected

in sheep

of a K'/Clred cells

system

and human red cells

swollen

circulation

SH groups

adult

system

in osmotically

The presence

(21)

cells,

in the putatively

operative

reduced

COMMUNICATIONS

activated

known to inhibit

A cotransport

concerning

of the LK gene.

group

cursor

shown).

cells

required

of a K+/Cl-

(19)

in duck red cells

tumor

ions

Furosemide

red cells

not

Cl-

2'disulfonic

Kt efflux.

and avian

RESEARCH

NEM reacts precursor

with

cells.

reported here,

system as the

HK

reticulocyte

concentration

to that

preof an

LK cell. This

work

was supported

by U.S.P.H.S.

grant

HL ZPOl-12,157.

References 1. 2. 3. 4.

Ellory, J.C. (1977) In: Membrane Transport in Red Cells. J.C. Ellory, V.L. Lew, Editors, Acad. Press. London, pp. 363-382. Lauf, P.K. (1979) In: Membrane Transport in Biology, Vol. I, G. Giebisch, D.C. Tosteson, and H.H. Ussing, editors. Springer Verl. New York-Heidelberg-Berlin, pp. 291-348. Evans, J.V. (1954) Nature 174, 931-932. Hoffman, P.G. and Tosteson, D.C. (1971) J. Gen. Physiol. 58, 438-466.

1427

Vol. 92. No. 4. 1980

5. F: a. 9.

10. 11. 12. 13. 14.

E: 17.

18. 19. ;:: 22. 23. 24.

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Ellory, J.C., Sachs, T.R., Dunham, P.B. and Hoffman, T.F. (1972) Biomembranes, Vol. 3, pp. 237-245, Plenum Press, New York. Joiner, C.H. and Lauf, P.K. (1975) J. Memb. Biol. 21, 99-112. Joiner, C.H. and Lauf, P.K. (1973) J. Physiol. (Lond) 282, 155-175. Tosteson, D.C. and Hoffman, J.F. (1960) J. Gen. Physiol. 44, 169-194. Rothstein, A. (1970) In: Current Topics in Membranes and Transport, Vol. I F. Bronner and A. Kleinzeller, editors, pp. 136-176. Kniuf, P.A. and Rothstein, A. (1971) J. Gen. Physiol. 58, 211-223. Garrahan, P.J. and Rega, A.F. (1967) J. Physiol. (Lond) 193, 459-466. Sachs, J.R., Ellory, J.C., Kropp, D.L., Dunham, P.B. and Hoffman, J.F. (1974) J. Gen. Physiol. 63, 389-414. Dick, D.A.T., Dick, E.G. and Tosteson, D.C. (1969) J. Gen. Physiol. 54, 123-133. Motais, R. and Sola, F. (1973) J. Physiol. (Lond) 233, 423-438. Lauf, P.K., Becker, B. and Duhm, T. (1979) The Physiologist, 22, 75. Lauf, P.K., Stiehl, B.J. and Joiner, C.H. (1977) J. Gen. Physiol. 70, 221-242. Gunn, R.B. (1979) In: Membrane Transport in Biology, Vol. II, G. Giebisch, Springer-Verlag, New York-HeidelD.C. Tosteson and H.H. Ussing, editors. berg, pp. 59-80. Wiley, J.S. and Cooper, R.A. (1974) J. Clin. Invest. 53, 745-755. McManus, T.J. and Schmidt, III, W.F. (1978) In: Membrane Transport ProRavens Press, New York, pp. 79-106. cesses, J.F. Hoffman, editor. Kregenow, F. and Caruk, T. (1979) The Physiologist 22, 73. C. and Pfeiffer, B. (1978) In: Cell MemGeck, P., Heinz, E., Pietrzyk, brane Receptors for Drugs and Hormones, A multidisciplinary approach. R.W. Straub, L. Bolis, editors. Ravens Press, pp. 301-307. G.W. and Ellory, J.C. (1980) Proc. Nat. Acad. Sci. Dunham, P.B., Stewart, (USA), ' Ellory,'!.!!rez% Dunham, P.B. (1980) In: Membrane Transport in Erythrocytes, Alfred Benzon Symposium 14, U. Lasser, H.H. Ussing, and J.O. in press, Munksgaard, Copenhagen. Wieth, editors. Kim, H.D., Theg, B.E. and Lauf, P.K. (1979) The Physiologist, 22, 69.

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