Morphological changes in asymmetric erythrocyte membranes induced by electrolytes

Vol. 70, No. 3,1976

BIOCHEMICAL

MORPHOLOGICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CHANGES IN ASYMMBTRIC

ERYTHROCYTE

MBMBRANES INDUCED BY ELECTROLYTES

Robert

M. Johnson

Departments

and Jennifer

of Biochemistry

Wayne State

Received

March

Besides

normal

(2)

interest.

Bessis

these

forms.

The echinocyte

(crenations), indented

cup-shaped

the

erythrocyte

that

the

shape

cyte

bilayer that Copyright AN rights

In

forces,

the

couple well-washed,

and,

as a normal

of the

red

tending

framework

two are covered the

normal

cell

depends

observations

of a recently

proposed

0 I976 by Academic Press, Inc. of reproduction in any form reserved.

that

on the

‘925

like

from

compounds

each class

This

either

that

either

these

balance

cell

out

into

disc.

These

erythrocyte

is an

(3) pointed

biconcave

the

permeable

spikes

stomatocyte

of an agent

VJe would

of particular

with

remarkably,

on a

(1) and

erythrocytes

to convert

hypothesis.

can take

classified

1968 Deuticke

combination

or to an echinocyte.

within

a sphere

converted

the

left

red cells

Among them,

or echinocytes,

were antagonistic:

disc,

a sea urchin:

cell.

agents

stomatocytes

48201

has recently

is

resembling

many chemical

opposing

biconcave

of shapes.

described

Michigan

School

human erythrocyte membranes are made permanently by EDTA. These ghosts can exhibit the stomatosequence of shape changes in response to the medium. The changes are instantaneous and observations can be explained on the basis of of cations on charged phospholipids together couple hypothesis.

the

range

Medical

24,1976

SUMMARY. Washed leaky to cations cyte-disc-echinocyte electrolytes in reversible. The the known effects with the bilayer

wide

and Physiology

University

Detroit,

Robinson

suggested of

two

to a stomato-

can be explained (4) theory, to report,

ghosts

the first,

can exhibit

the

Vol. 70, No. 3, 1976

full

range

simply

BIOCHEMICAL

of the

stomatocyte-disc-echinocyte

in response

tions

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to cations,

are consistent

with

and,

shape spectrum

secondly,

the bilayer

that

couple

the observa-

hypothesis.

ghosts are preMATERIALS AND METHODS. Washed hemoglobin-free For pared as previously described (5) and used within 3-4 h. photography, a wet mount of a ghost suspension (log/ml) was left undisturbed in the cold for 20-30 minutes to permit the ghosts to settle. They were then photographed using Nomarski optics. Distilled freshly deionized water free of Ca and Mg by flame All chemicals are reagent grade. photometry was used for buffers. RESULTS. during

The ghosts

their

to render for

preparation,

the conditions

be maintained responsive

the

across to the

final

ghosts into

that

effects

osmotic

the

levels

crenation tions

ghosts

ghost

the buffer

with

(S,7).

echinocytes are not

into

(Fig.

They are not

morphology medium.

crenated.

in crenating

is nevertheless Figure

la which

stomatocytes

discs

In order Figure

of can

pH 7.4,

biconcave

operating,

recover

gradient

concentration

lc).

Under

presence

are mostly

electrolyte

was proven

do not

5 mM Tris-Cl,

The ghosts

(6,7)

is (Fig.

inlb)

to demonstrate

Id shows the ghosts KC1 and Na acetate

NaCl,

ghosts.

cations are also crenating agents. Table I shows ++ ++ ++ and Sr that bring ribout complete of Ca I Mg

of ghosts

are more

shows that

buffer.

effective

Divalent

of

are converted

in 250 mM sucrose.

methods

or chemical

these membranes,

in 7 mM NaCl,

and finally,

are equally

no osmotic

As the univalent

the

these

This

to 22Va _ , even in the

composition

washing

and spheres. creased,

microscopy,

Although

is reported

to cations.

described

impermeability

shows the ghosts

treatment

by previously

cations.

in 1 mPI EDTA, pH 7.4,

permeable

we use for

any appreciable

is

and this

them permanently

our preparation

divalent

are washed twice

effective

anions

It

in 7 mM NaCl.

are not

than

univalent.

effective.

is clear

that

In addition, The trivalent

926

divalent Table cation,

caI

Vol. 70, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a.

Figure

1.

Nomarski phase micrcgraphs of permeable hemoglobinfree human erythrocyte membranes. All solutions are buffered with 5 mM Tris, pH 7.4 (a) in 7 mM NaCl; (b) in 25 mP4NaCl; (c) in 75 mM NaCl; (d) in 250 mM sucrose. 927

BIOCHEMICAL

Vol. 70, No. 3, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

I

Wet mounts of ghosts were observed by phase microscopy immediately after being suspended in solutions of 5 mM Tris, 7 mM NaCl, pH 7 with electrolyte concentrations ranging of electrolyte which from lo-6 to lo- -4 M. The concentration gave essentially complete crenation is listed. mM

NaCl KC1 Na acetate Na2S04 sodium phosphate Mg Cl2 Ca Cl2 Sr Cl2

+++ La , caused aggregation No changes

were

Although after

the

are

of ghost ghosts

were

prepared

echinocyte

spectrum membrane

proposed,

based

erythrocyte in

the

leaflet

outer

properties.

while

the

inner

various

the leaflet

can affect

to affect (8),

the effects

The method

the

results,

since

respond

in

in

stomatocyte-discfact

depends

explanation

are asymmetrically lipids while

leaflet

the

(a) The distributed located

other

phospholipids,

inner,

cytoplasmic,

has a net negative

of charged 928

are

charge

charge. phospholipids.

on

can be

of phospholipids.

compose

the

buffer.

morphology

bilayer,

packing

hour

the

concentrations.

the

has a net the

All

A plausible

outer

l/2

solutions,

immediately.

The choline-containing of the

10 -5 M.

than

1 were taken

that

phospholipids

phosphatidylserine, As a result,

trolytes

demonstrate cell

greater

pH 8.0

on known properties

9).

let.

appear

in Tris

of red

membrane

(reviewed

notably

prepared

The results

intrinsic

salt

5 mM phosphate,

same way as ghosts DISCUSSION.

in the

the

does not in

40 50 1 1 1

pH 7.4

Figure

can be seen

by changing

isolation

in

suspended

changes

reversible

buffer,

photographs

ghosts

morphological

100

at concentrations -5 I1 La +++ 10 .

seen with

the

100 100

of

on

leafzero,

(b) ElecFor

BIOCHEMICAL

Vol. 70, No. 3, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

++ example, when phosphatidylserine monolayers are exposed to Ca ++ presumably by or Mg , the average area per head group declines, a reduction bilayers

made with

reduce This

of charge-charge

charge

negatively

repulsion

was shown (11)

cations)

promote

the

of

acidic

lipid

of the

of

tion

is

outer

tighter

leaflets

convert

in the

to

positively

negative

inner

anethetics

entially

enter

changes

stomatocytes charged

leaflet,

crenate

the outer

required

Taken together, that

an increase

both

sides

of

the

about

cell.

On the other

reduced,

the

the outer stomatocyte.

with

this

statements

area of the

uncharged

leaflet,

if inner

of

inner as a

concentra-

the

inner

and

Cationic

anesthetics

Sheets

and Singer

its (3);

these

to yield

At some intermediate

929

drugs

prefer-

area ratio

changes were

picture. lead

to the prediction in a huffer diminish

mechanism,

leaflet

insert

of the

preferentially

electrolyte

(4)

Similarly,

area.

concentration

by a shielding hand,

the areas

has

the

diminish,

the morphological

in electrolyte

leaflet,

electrolytes

Calculations

of the membrane will

inner

of

compounds preferentially

leaflet.

these

fluid

potential

as electrolyte

increasing

made (4) and are consistent

packed

surface

(3).

erythrocytes

to bring

(pII and

at constant

membrane will

morphology.

that

phospholipids.

the area of

packing,

ghost

loosely

by univalent

of

factors

of the

phase,

Therefore,

in

such factors

in the

(c) The ratio

affects

that

anionic

(12).

erythrocyte

erythrocytes

proposed

packed ordered

phospholipid

increased.

that

from the

phospholipids

measured

result

packing

a reduction

been directly leaflet

closer

transition

Similarly,

phospholipids,

by demonstrating

Moreover,

monolayers

(10).

charged

favor

phase to the more tightly temperature.

repulsion

expand

a spherical

concentration,

the

and crenate

concentrations should

bathing area the

are relative

to

indented the ghost

will

BIOCHEMICAL

Vol. 70, No. 3, 1976

be a biconcave

may

addition,

help

explain

the

they

sulting

tend

In the

yield

presence (Table

right-side This

may

that

will

it

changes actin

the

other proposed cations

and

evaqinated This

re-

may be of general

these

can exhibit

membranes

sequence. are

shape

all

it

the cell

is unlikely

spectrin-

shape

bilayer. can respond

changes the

full

If

the

asymmetrically

changes

shape

the morphological

For example,

membranes

shape

for

in

the

(14)

to small

changes

are reversible, range

of the

and stomato-

phospholipids

of

distributed,

the

presence

of divalent

for technical Health Service

REFERENCES 1. 2. 3.

(Bessis, Weed and Bessis, M. (1973) in "Red Cell Shape" Leblond, eds.), Springer Verlaq, New York, pp. l-25. Bessis, M. and Weed, R.I. (1973) Adv. Biol. Med Phys. 14, 35-91. Deuticke, B. (1968) Biochem. Biophys. Acta 163, 494-500.

930

and

Nevertheless

importance.

We thank Mr. Steven Sucech work was supported by Public

to

(13).

observed since

phospholipid

that

for

the

in controlling

the

concentration, ghost

for

ghosts

be expected

observed

modification,

role

some

isolated

ACKNOWLEDGEMENTS. assistance. This Grant HL-15793.

la);

the

however,

is also

here

membranes.

shown that

mechanism

disruption,

and would

to account

with

biological

(Fig.

stomotocytes

the

vesicles.

cyte-disc-echinocyte

Without

cations,

be sufficient

isolated

are

for

of divalent

interacts

in cation

vesicles.

(13)

as observed

subsequent

plays

procedures

Upon ghost

advanced

require

complex

we have

that

out

observations

to be inside-out,

of erythrocyte

presumably

buffers

our

be expected

I)

explanation

changes

of Steck's

invaqinate.

would

are crenated

that

and inside-out dilute

to

vesicle

(3).

in

what we observe.

propose

selectivity

right-side-out

Mq or Ca, ghosts i.e.,

is exactly

we tentatively

In

generating

This

disc.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 70, No. 3, 1976

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Sheets, M.P. and Singer, S.J. (1974) Proc. Nat. Acad. Sci. 2, 4457-4461. Johnson, R.M. (1975) J. Membrane Biol. 21, 273-289. Hoffman, J.F. (1962) Circulation 26, 1201-1213. Bodemann, H. and Passow, II. (1972) J. Membrane Biol. S, l-26. Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochem. 10, 2606-2617. Bretscher, M.S. and Raff, M.C. (1975) Nature -'258 43-49. Papahadjopoulos, D. (1968) Biochem. Biophys Acta 163, 240-254. H. and Eibl, H. (1974) Proc. Nat. Acad. Sci. Trguble, 71, 214-219. McDonald, R.C. and Bangham, A.D. (1972) J. Membrane Biol. I, 29-53. (1972) Nature New Eiol. Kant, J.A. and Steck, T.L. -'240 26-27. LUX, S.E. and John, K.M. (1975) Blood -'46 1052.

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