Arachidonate metabolite(s) increase the permeability of the plasma membrane of the neutrophils to calcium

Vol.

92, No.

February

BIOCHEMICAL

4. 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

27, 1980

ARACHIDONATE

METABOLITE(S)

M. Departments

Received

of

INCREASE THE OF THE NEUTROPHILS

Volpi,

P.H.

Physiology Center,

November

PERMEABILITY TO CALCIUM

Naccache

and

and Pathology, Farmington,

OF THE

R.I.

1231-1237

PLASMA

MEMBRANE

Sha'afi

University Connecticut

of Connecticut 06032

Health

6.1979

SUMMARY: The relationship between arachidonate metabolism and stimulated calThe addition cium fluxes in rabbit peritoneal neutrophils has been investigated. of arachidonate to the neutrophils was found to cause a rapid and significant increase in the permeability of the plasma membrane to calcium. This effect is specific to calcium, concentration dependent and sensitive to inhibitors of the lipoxygenase mediated metabolic pathway(s). These results strongly suggest that arachidonate metabolites are directly involved in the mechanisms underlying calcium gating in the neutrophils.

INTRODUCTION

The in

the

possibility

that

initiation

and

tactic

factors

and

recent

studies

(l-8).

genase

pathway

have

(1)

activity

logical

for

nate

metabolism

the

lipoxygenase

the

effects

most

on

and

plasma the

various such

tie

now

wish

by

to

Abbreviations: 8,10,14-eiccsatetraenoic phen?;lalanine:

are to

pathway(s) are of

some

to

by

the

ability

(3).

This

which

are

fluxes

the

Nordihydroguaiaretic acid: F-Met-Leu-Phe.

results

and

secretory

acid: 12-HETE;

experiments

in

suggested this

fatty to

affected

NDGA: l?-L-hydroxy-5, Formyl-methionyl-leucyl-

bio-

inhibit

conclusion

which

the

arachido-

was by

the

that acid

or

alter based

chemotactic

(F-Met-Leu-Phe) of

of

lipoxy-

of

intermediate

formyl-methionyl-leucyl-phenylalanine describe

the

specifically

of

chemo-

results

of

recently

to

the

aspects

which

generated

calcium

(8)

involved by

of

inhibitors

lipoxygenase calcium

by products

We have

be

neutrophils

these

compounds

mediated

the

chemotactic

sensitive

(1,3).

permeability of

and

addition,

those

might

strengthened

both

In

particularly

that

been

possess

arachidonate

parameters as

to

metabolites of

arachidonate

neutrophils.

arachidonate

membrane

factors

shown

its

activation

has

example,

mediated of

the

stimuli

For been

and/or of

secretory

of

probably

the

regulation

rabbit

activities

arachidonate

(3). above

hypothesis.

Vol.

92, No.

namely

4, 1980

the

membrane

BIOCHEMICAL

ability

or

AND

arachidonate

permeability

to

BIOPHYSICAL

and/or

calcium,

was

metabolites

directly

MATERIALS

RESEARCH

AND

to

COMMUNICATIONS

increase

the

plasma

examined.

METHODS

Rabbit peritoneal neutrophils were collected and handled as previously described (11). The concentration of CaC12 in the Hanks' balanced salt solution used here and previously (3) was adjusted to 0.5 mM. Magnesium was omitted in order to minimize spontaneous and arachidonate induced cellaggregation (12,13). Protein was omitted from the incubation medium. Lactate dehydrogenase release, a measure of cell death, was minimal (15%) under all of the conditions tested. Calcium transport was method previously described at 37°C preceeded all further the cells two minutes before Stock solutions dimethyl sulfoxide with fresh batches

measured using the rapid sampling, in detail (14,15). A 20 minutes experimental manipulations. 45Ca and/or arachidonic acid.

of arachidonic and stored at or arachidonic

acid

Arachidonic Sigma Chem. Co., (Boston, Ma.) as

and St. Louis, CaC12 in

acid and NDGA were -70°C under nitrogen. acid were routinely

Nordihydroguaiaretic acid MO. 45Ca was obtained water. All other reagents

RESULTS The

essential

concerning latter, for

as the

feature

the

intracellular

yet

unidentified

previously

permeability

of

the

membrane the

directly

rate

45 Ca

influx

in

our

enhances

the

observed

within

stimulation this

initial but

working rate

measurable

45Ca first

45 Ca influx

concentration

rates

of

of

45 Ca

at

into

exogenous

minute

after

shown of

the

in

from

to

persist

in

dependence

of

in

calcium

(14,16). on

1,

This

at

the

and

re-

greatly effect

arachidonate least

the

as

arachidonate

of

the

responsible

Figure

5x10m6M

for

that

arachidonate

addition

to

is

changes

neutrophils.

the

postulated

part,

induced

of

addition

recently

least

neutrophils

As

appears

have

the

effect

influx

in

(NDGA) were purchased from New England Nuclear were analytical grade.

metabolites

factor of

the

at lO-lM experiments

can

be

and

the

10 minutes

at

arachidonate.

2 illustrates of

are,

neutrophils.

scheme,

the of

Figure

the

we

arachidonate

chemotactic

plasma

tested

by

which

compounds,

thus

quired

of

made up Control performed.

to

DISCUSSION

model

role

We have of

the

described

of

AND

silicone oil preincubation NDGA was added

the influx

enhancements

on of

the 45 Ca

the

magnitude

concentration influx

1232

can

of be

of

the

arachidonate.

observed

at

increase Small

concentrations

in

Vol.

92, No.

BIOCHEMICAL

4, 1980

x--x -

AND

CONTROL 5 x 10.6M

BIOPHYSICAL

ARACHIDONIC

RESEARCH

ACIG

5

10

TIME Figure

of

1:

arachidonate

level

off

below at

centrations (1)

and

nate

enhanced

a net

experiments the the

influx

of

calcium

greatly

effect

calcium

in

of

rates in

that

not

shown).

of

arachidonate neither

are

thus

here

we

in

excess

45

Ca

of

neutrophils.

the

effect

on the

if

in

addition

not

rates

of

1233

The

conrelease

identical.

here

that and

are

addition

thus

the

or

that for

cytochalasin rate

6

of

permeability 42K

arachido-

responsible of

on

of

to

enzyme

calcium

membrane influx

10W5M.

observed

arachidonate

plasma

appears

lysosomal

described

of

the

(8),

The

Ca influx into the added at zero time.

of

exchangeable

influx

45

stimulation

similar

have

the

of

the

chemotaxis

level of

of

arachidonate

influx shown

on the rate of and/or 45Ca were

magnitude

causing

enhances

(results The

The

steady-state

in

influx

to

not

increase

ug/ml)

10m7M.

calcium

(MINUTES)

arachidonate Arachidonate

arachidonate

increases

the

added

concentrations of

In

(5

Effect of neutrophils.

COMMUNICATIONS

l4 C-urea

45Ca

is

specific were

Vol.

92, No.

4, 1980

BIOCHEMICAL

AND

7

8

-LOG Figure

found

to

be

not

shown).

were

also

up

to

2:

altered

under

ruled

as

causes to

to

centage

(less

respond

to

added

similar

to

that

than

(17)

arachidonate

(6)

The

previously

101)

of

and

or

more

the

plasma

human

red

cells

(results

not

an

ability

as

found

shown).

not In

in

studies the

in

lipid the to

were

45 Ca

in

influx,

which

neutrophils

(results bilayer

addition

the to

of

affect

addition,

preparations

increase

of

Ca influx

membrane

effects was

neutrophils by

45

increased

observed

the

the

were

of

reported

these a small

found

not

perto

a variability functional

re-

metabolize

exogeneous

examined. published

induced

itself

which

the

arachidonate

F-Met-Leu-Phe

one

5

ACING

for

calcium

previously

sponsiveness

rather

conditions

arachidonate

arachidonate

COMMUNICATIONS

6

~~RA~HIDONI~

alterations

out

cells'permeability

on

RESEARCH

Concentr ion dependence of the effect of arachidonate on the rate of sE Ca influx into the neutrophils. The data were generated from experiments such as the one depicted in figure 1 at the 5 minutes time point. Each point represent the average of at least two experiments each carried out in duplicates.

Nonspecific

lo-bM

BIOPHYSICAL

calcium

which of

experiments

its

is

dealing

fluxes

(3)

responsible

for

strongly

the

We have

metabolites.

1234

with

the

effects

suggest

that

permeability thus

examined

of it

changes the

NDGA is

not

but

sensitivity

Vol.

92,

No.

4,

BIOCHEMICAL

1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

200-

X 3 ii Z-

100,

CONTROL

Figure

of

the

3:

arachidonate

action

of

ments

are

increases effects

the

the

rate

initial

arachidonate

of The

trations neutrophil

in

larger

results of

functions

3.

of

45Ca

inhibitions

,O%M NDGA

(chemotaxis

The

the

lower

at

thus have and

in

yet

clearly previously lysosomal

1235

to

of

experi-

5 x

these

10T6

and

the

for

an

unidentified

enzyme

of

inhibition

that

shown release)

to

the

NDGA

arachidonate.

metabolites

demonstrate been

of

presence of

the

10m5M

inhibitions

concentration evidence

as

neutrophils

results

demonstrated

into

which

rabbit

Significant

preliminary

above

the

influx.

at

conversion described

AA IO-5M

Arachidonate

clearly

obtained

arachidonate

in NDGA.

Figure

are

addition,

arachidonate

influx

inhibitor in

somewhat

We have,

45Ca

1iPOXYgenaSe

AA 5xl0‘6M NDGA

45 Ca influx. on control and arachidonate stimulated 2 minutes prior to 45Ca and/or arachidonate. Each point represents the 5 minute time points. least two experiments each carried out in duplicates. the concentration of NDGA was 10-5 M. Arachidonate indicated concentrations.

induced

illustrated

of

with

NDGA

Effect of NDGA NDGA was added Data based on average of at Whenever used, was used at the

NDGA AA IO-SM 5Xl0’5M

by (2).

conceninduce increase,

various

Vol.

92, No.

in

an

4, 1980

NDGA

sensitive

neutrophils

to

necessary

as

manner,

calcium.

requirement

activation from

BIOCHEMICAL

by

results

the

previously

chemotactic

factors

followed

unknown,

by

its

of

are

the

thus

which

the

directly

liberation the

involved

membrane

consistent model

by

COMMUNICATIONS

plasma

both

conversion

is

RESEARCH

postulated

in

metabolite(s)

BIOPHYSICAL

permeability

These for

phospholipids yet

the

AND

with neutrophil

of

arachidonate

calcium

the

and

of

lipoxygenase in

of

a

to

some,

(21)

in

was

found

gating

mechanisms. In which to

conjunction

12-HETE, be rapidly

addition,

permeability

generated

is

next

membrane

mediating

its

cellular here

is

role

in

of

the

monally

previously induced

This

study

(3) target

was

gating

of

gating

calcium

supported

thus

described

membrane

a neutrophil

activity

The

elucidation

of

most

probably

await view the

relevant (22).

National

Institute

of

towards the the the

biochemical generalized presented

a variety

of

the

mechanisms

hypothesis to

mechanisms

of

Health

hor-

grant

AI 13734.

REFERENCES

1.

2. 3. 4.

Naccache, P.H., Showell, H.J., Becker, E.L. and Sha'afi, R.I. 1979. Biochem. Biophys. Res. Comm. 87, 282-299. Showell, H.J., Naccache, P.H., Sha'afi, R.I., Walenga, R., Feinstein, 1.l.B. and Becker, E.L. 1979. J. Cell Biol. (In press). liaccache, P.H., Showell, H.J., Becker, E.L. and Sha'afi, R.I. 1979. Biochem. Biophys. Res. Comm. 89, 1224-1230. Borgeat, P., Hamberg, M. and Samuelsson, B. 1976. J. Biol. Chem. 251, 7816-7820.

1236

in

(18-20).

required

mechanisms,

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the

be directly

part

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In

may,

phospholipase

homogenates

involved.

activation

in

hypothesis

demonstration

will

thus

above

towards

calcium.

metabolite

cell

the

with

Parker

activity,

neutrophil

direct

and

chemotactic

previously

from

to

may

Stenson

activity

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calcium

distribution and

the

metabolite(s)

permeability

identification

for

recovered

required

arachidonate

plasma

with

increasing

been

of

phospholipids,

a basis

has

report

product into

provide

which

What

recent

a lipoxygenase

help

E.Coli

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BIOCHEMICAL

AND

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RESEARCH

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Borgeat, P. and Samuelsson, B. 1979. Proc. Nat'l. Acad. Sci. USA. 76. 3213-3217. B%rgeat, P. and Samuelsson, B. 1979. Proc. Natl. Acad. Sci. USA. 76, 2148-2152. Hirata, F., Carcoran, B.A., Venkatasubramanian, K., Schiffmann, E. 1979. Proc. Natl. Acad. Sci. USA. 76, 2640-2643. and Axelrod, J. Turner, S.R. and Lynn, W.S. 1978. In Leukocyte Chemotaxis, Gallin, 'J.1. and Quie, P.G. (Eds.). Raven Press, New York, 289-300. Tappel, A.L., Lundberg, W.O. and Boyer, P.D. 1953. Arch. Biochem. Biophys. 42, 293-304. Hanberg, M. 1976. Biochim. Biophys. Acta. 431, 651-654. Showell, H.J., Williams, P., Becker, E.L., Naccache, P.H. and Sha'afi, R.I. 1979. J. Reticul. Endo. Sot. 3, 139-150. O'Flaherty, J.T., Kreutzer, D.L. and Ward, P.A. 1977. 3. Immunol. 119, 232-239. O'Flaherty, J.T., Showell, H.J., Becker, E.L., and Ward, P.A. 1979. Am. J. Pathol. 95, 433-444. Naccache, P.H., Showell, H.J., Becker, E.L. and Sha'afi, R.I. 1977. J. Cell Biol. 73, 428-444. Naccache, P.H., Showell, H.J., Becker, E.L. and Sha'afi, R.I. 1979. J. Cell Physiol. loo, 233-250. Gallin, J.I. and Rosenthal, A.S. 1974. J. Cell Biol. 62, 594-609. Showell, H.J., Naccache, P.H., Becker, E.L. and Sha'afi, R.I. 1979. Fed. Proc. 2, 1023. Beckerdite, S., Mooney, C., Weiss, J., Franson, R. and Elsbach, P. 1974. J. Exp. Med. 140, 396-409. Weiss, J., Franson, R., Beckerdite, S., Schmeidler, K. and Elsbach, P. 1975. J. Clin. Invest. 55, 33-42. Weiss, J. and Elsbach, P. 1977. Biochim. Biophys. Acta. 466, 23-33. Stenson, W.F., and Parker C.W. 1979. Prostaglandins 18, 285-292. Putney, J.W. 1979. Pharmacol. Rev. 30, 209-245.

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