Calcium mobilization in fluoride activated human neutrophils

Vol.

133,

No.

November

1, 1985

27,

BIOCHEMICAL

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RESEARCH

Pages

1985

CALCIUM MXBILIZATICN

IN

and Kenneth Wng

Departments of Pharmacology and Medicine of Alberta, Edmonton, Alberta, T6G 2G3 CANADA

University October

161-167

FLUORIDE ACTIVATED HUMAN -PHILS

F. Strnad

Colette

Received

BIOPHYSICAL

AND

7, 1985

Fluoride ion, at concentrations above 10 mM, was faclnd to elicit a rise in intracellillar calcium levels in neutrwhils, as mnitored by changes in @in 2 fluorescence intensity. The ca1ci.m mobilization response was characterized by a lag period of 4 to 10 min. and a prolonged duration of action (greater than 20 min.). In contrast, the chemotactic peptide, formylmethionyl-leucyl-phenylalanine, induced a rise in intracellular calcium Preinctition of the cells with 1 concentrations which peaked within 1 min. pg/ml pertzsis toxin resulted in inhibition of the fonnylrrethionyl-leucylphenylalanine induced response, but not that mediated 13y fltioride. Recent evidence suggests that the fotmylmzthionyl-leucyl-phenylalanine receptor is coupled to phospholipase C and phcephoinasitide degradation thrmgh a guanine nucleotide binding protein susceptible to inhibition &J pertussis toxin. Present results suggest that fliloride ion may serve to activate this protein in a manner resistant to inhibition b perttissis toxin. 0 1985 Academic Press, Inc. SUMMARY:

Althcugh

the ability

human nedtrophils mechanism

for

propceed. role (4) for

has the

which

serve

CAMP levels

Recent

action

reports

binding

proteins

calcium

mobilization agonists

and

units

inhibitory

of

Ni

2,

gclanine

betwzen

has

yet

binding

been

proteins

and the receptors

cyclase

Hover,

respectively.

(5,

in

has a wzll-established

are associated txlrst

ion

nucleotide

the

hrst

31, no satisfactory

halide

fluoride

hormOnes,

in the neutrophil

a respiratory

(1, this

system,

N,

of the respiratory

of fluoride

previously

cyclase

as intermediary

than activation,

dependent

inflionmatory

and

ion to activate

reported

of both the

stimillatoq

mediator

been

In the adenylate

as an activator

elevated

of fluoride

with

since

suppression,

rather

61, cAMP is unlikely

to be the

action. have

implicated

in the transdilction as a mans

the

involvement

of thoee hormonal

of celliilar

such as the chemotactic

161

activation peptide,

All

of quanine signals

(7,

nucleotide

which 8, 9).

exploit Receptor

formylmethionyl-leucyl-

Copyright 0 198.5 rights of reproduction

0006-291X/85 $1.50 by Academic Press, Inc. in any form reserved.

Vol.

133,

phenylalanine well

BIOCHEMICAL

No. 1, 1985

(Fhlet-Leu-Phe),

as the release

have

mobilization inhibited toxin

of

with

calcium

by treatment

elaborated

suggested phcspholipase

of cells

inositol

and granular of

Notably,

the

with

for

for

the activator

1,4,5-trisphcsphate,

responsible

toxin

12, 13, 14),

factor

C, the enzyme responsible

a

calcium

mobilization

of Ni (15).

fran

an AB

receptors

and

of phosphoinositides

kinase

the

all

Ni has been

agonist

degradation

soluble

are

the ADPribcsylation

beteen

of protein

water

and the

reactions

(11,

as

These responses

preceding

which catalyzes

as a coupling

aggregation,

and

polyphosphoinositides

pertussis

pertussis,

COMMUNICATIONS

enzymes.

of the 41,000 MW subunit

14) to act

1,2-diacylglycerol,

into

RESEARCH

chemotaxis

turnover

(10).

inactivation

(13,

(02-)

the

by Bordetella

and concanitant

BIOPHYSICAL

induce neutrophil

of superoxide

been correlated

AND

C (rev.

product

in 16), and

believed

endoplasmic

to

reticulun

be

(rev.

in 9). In view binding

of

fluoride's

proteins,

activation

we have examined

of the respiratory

this

ware the case,

of

"calcium-mobilizing

fluoride

degradation

calcium

concentrations.

The present

of

data sh-

free

calciinn

levels,

The

calcium

mobilization

superoxide action.

elicits

production This

a respiratory

Hurst

the

that

possibility

may be occurring to

at the level

mimic

an increase

response

is

induced

by the fluorescent paralleled

a prolonged consistent

that

lag period with

probe,

fluoride

cytasolic

that of

ain

2.

activated

and sustained

activation

the

in intracellular

the hypothesis

b means of persistent

C with of

calcium of

If

Ni.

of agonists

phcspholipase

that

induced of

the action

and elevation

fluoride

nucleotide

fluoride

pslyphosphoinoeitides

in having

burst

of guanine

by activating

as monitored

finding

as an activator

would be expected

receptors"

resultant

of

efficacy

&ration fluoride

Ni*

METHODSANDMATERIALS The following materials ware purchased fran the sources indicated: 2/AM fran Calbiochem (San Diego, CA), pert;lssis toxin (islet activating protein) fran List Biological Laboratories (Campbell, CA), Formyl-methionylleucyl-phenylalanine (Fmat-Leu-Phe) fran Sigma Chemical (St. Lais, MD), and sodisn fluoride fran Fisher Scientific Co. (Pittsburgh, PA). Human neutrophils were isolated as previously described (6) and suspended in Hanks' Balanced Salt Solution (HBSS), pH 7.4 and 37' C, at a density of

Qdin

162

Vol.

133.

No.

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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lo6 cells/ml. Intracellillar calciian levels were monitored by the Quin 2 fluorescence technique established by Tsien et al. (17). Cell ware separated into two grmps, one of which was incubated with 1 vg/ml of pertilssis toxin, while the other was left untreated. After 30 min., Quin 2/AM, at a final concentration of 10 uM, was added to each cell suspension and the incubation was continued for another 30 min. At the end of the total 1 bar incubation and washed once in EBBS, then resuspended at a time, cells were centrifuged concentration of 2 x lo6 cells/ml for fluorescence measurerrents. The latter ware conducted in a Perkin Elner Model MKF-4 fluorescence spectrophotarteter, thermostatically controlled at 37' C. The excitation and emission wavelengths for the fluorescence measurements were 339 nm and 492 rm with 5 run and 15 run Maximal and minimal fluorescence signals wzre obtained slits, respectively. by lysing the Quin 2 loaded cells with Triton X-100 (1%) in the presence of The plateau for fluoride either 1.6 mM calciun (Fma ) or 2 mM WA (Fmin). mediated calcium mbiliza t. ion typically lay about half-way between the baseline fluorescence for unstimulated cells and the Fmax value. 10 X

RESULTS AND DISCUSSION Upon exposure an increase

in cytcsolic

of the fluorescent

reflect the

calcium

the time interval

fluoride

and acceleration dependent

that

induces

a manner which

(Ca2+) i

probe,

is

not

duration

for

readily

of

reversible

Ni

(18).

lag period

is

into

of

its

induced

calcium

its

site

of action

consistent

with

the fact

35~ and 4lK &ydnits

in

of the lag interval

ware observed The time

concomitant

in

response,

to be dose-

course

superoxide

of the release

Relationship of Ca*+ mobilization to NaF concentration in a 2 x 106 -muspension of neutrcphils. 'Ibe arrow indicates the time of addition 'These results depict the tracings obtained fran a single of stimiilils. experiment which is representative of at least five experiments. 163

use

of 4-10 min which may

Reduction

,of 18 r&J. the

thrcxlgh

evoked calcium

mobilization

concentration that

Fluoride

of the fluoride

of the rate of calcium

paralleled

1).

by Fmet-Leu-Phe,

respond with

as monitored

the ion to reach

the dissociation

up to an optimal increase

a prolonged

required

elicited

(Ca'+)i,

kvek

Quin 2 (Fig.

b

The extended

membrane.

as compared with that

free calcia

is characterized

mobilization

of NaF, human neutrcphils

to mM concentrations

Vol.

133,

BIOCHEMICAL

No. 1, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

w

Ca2+ mobilization & 1 PM Fn'et-Leu-Phe (A and B) and 18 mMNaF (C In the presence (B and D) and absence (A and C) of pretreatment with 1 pg/ml pertussis toxin. The arro& indicate the tti of addition of stimulus. These results depict the tracings obtained fron a single experiment which is representative of at least three expzrinents.

reaction

(3).

At concentrations

response

appeared

been observed

evoked

rise

prcduction

to decline

in (lo),

the

.,;'fered

(maximal

respiratory

burst responses

Fmat-I,eu-Phe

rise

(3).

toxin well

inhibits

as

the

of guanine

elicited

within

50%), and rapid

a phencmenon which

pathway.

that

response

stimulated

induced

the theory

fran

Fn-et-Leu-Phe

as

21,

transduction

by fluoride

15

set)

termination

The canparative

(l-2

of

superoxide

binding induced

smaller

min).

calcium

magnitude

The time curse

with

that

as a result

that

fluoride

of the

peptide

desensitization

Ni

(by

of the associated

of the chemotactic or

protein

in being of more rapid

(191,

brevity

inactivation

associated

F'mat-Leu-Phe

in (Ca2+ )i correlates

and/or

has also

the Wt-Leu-Phe

nucleotide

may be due to down-regulation

receptor

the Ca2+ mobilization

(2).

pertussis

(Fig.

reinforcing

mobilization

approximately

(12),

(Ca2+) i

in

not shckJn),

production

previmsly

involvenent

onset

(data

for supsroxide

As reported

of 20 mM and above,

of the

of GT~ hydrolysis

(4). Results

illustrated

mobilization its

was resistant

mechanism

of

Ni

prostaglandin unaffected

did

observe

2 shm

to inhibition is

is consistent

El stimulated @J pertussis

(21)

Fig.

activaticxl

Such a finding

protein.

Gilman

in

aderylate

toxin

(18,

pertussis

by pertussis unaffected with

toxin 164

toxin,

that

in the presence

It should inhibition

that of

the

inhibition

of

of fluoride

is

be noted that of

calcium

suggesting

ADP-ribosylation

the otservation

cyclase 20).

&

activated

fluoride

Rokoch and stimulated

Vol.

133,

No.

1, 1985

arachidonic

BIOCHEMICAL

acid

release.

accented

for

by the

nucleotide

binding

A2 dependent proteins Indeed, fran in

the

case

specific

profile acid

concentrations

in cyclic

neiltrophil

activation,

CAMP or adenylate does not result knxn

to

(5, 6).

responses

is

inhibitory process

prior

activatiton

our previous

unaffected

effect

finding

that

by the results

of Katada and co-workers

increasing aderylate to inhibit

the

adehylate

concentrations cyclase

activity.

prostaglandin

at

cyclase of

Since marked

at which

at

calcia

to autoinhibition,

as well

N,

the nedtrcphil

Indeed,

ftdoride

than the

first

High concentrations El stimulation

N,,

of

step

are

nedtrcphil

of neutrcphil

indicates

that

the

in the transduction

above 10 mM, fluoride unit.

This is suggested a biphasic

platelet

increasing, of fluoride

of adenylate 165

for

dibtyryl

CAMP levels

induced

(3)

(20) who observed

fluoride

the

phcsphorylation.

concentrations

activity

with

activation

at scm

(31,

of the E series,

elevated

agonists

as the Ni

responsible

of CAMP levels

F'mat-Leu-Phe

that

Nit rather

the

only

be considered

by means of receptor

appears

was

such as prcstaglandins

potentiate,

stimulates

on

in

elevation

per se.

p?Zferehtially

fluoride

cannot

of CAMP may be occurring

it

latter.

subject

of the

by CAMP or cyclase

to Nir possibly

Furthermore,

levels

than

differed

being 18 mu

the

concentrations

CAMP levels

agonists,

rather

activation.

response

of

these

two phenanana.

as experimental

inhibit,

fltioride

toxin

as an activator

nucleotide

cyclase

case

ware already

of elevating

in cell

responses

these

toxin

concentrations

by pertussis

proc&tion

serves

capable

alteration

the

guanine

C and phcspholipase

to

optimal

may be

Ni-like

by pertussis

of 20 mM and above,

fluoride

and is

with

effects

of

for the calciun-superoxide

release

to correlate

Althcugh

subsets

sensitivity

and 50 ~$4 in

and superoxide

COMMUNICATIONS

toxin

to the phcepholipase

release,

former,

arachidonate

is difficult

in partussis

distinct

a differential

of the

mobilization

of

RESEARCH

Upon ADPribosylation

exhibit

of

fluoride

unit,

proteins

of arachidonic

inhibition

it

coexistence

the dose-response

that

BIOPHYSICAL

The discrepancy

pathways.

might

AND

cyclase.

effect

membranes then wzre

of with

decreasing, also

fmnd

Together

these

Vol.

133,

BIOCHEMICAL

No. 1, 1985

observations

implicate

neutrophil

calcia

we propose

concentrations

prolonged

lead

to

persistent of

into

protein

the

mobilizer,

turnover

site

RESEARCH

of fluoride

we

COMMUNICATIONS

interaction

C activator,

1,4,5

trisphosphate.

in the presence

presently

during

unit.

cytosolic

elevate

are consequences Activation

C,

with

phcephatidylinositol

In an effort

of Ni would the

resultant

and the to further

studies

of

4,5-bisphcsphate

1,2-diacylglycerol,

conducting

of fluoride

to

burst

phospholipase

substrate,

kinase

are

ability

a respiratory

of

stimulation

inositol

fluoride's

of the Ni transducing

the principal

proposal,

that

and activate

stimulation

breakdcrwn

this

as the molecular

Ni

BIOPHYSICAL

stimulation.

In conclusion,

its

AND

of

calcium

substantiate

phosphoincsitide

in the neutrcphil.

ACKNOWLEIXMENTS This

work

was supported

Medical

Research

authors

would

the their

Odin

2

Council

like

to thank

techniqe

assistance

by Alberta

of Canada,

Heritage

Medical

and Arthritis

Society

Dr. E. McCoy for his helpful

and Carol

in the collection

Johnson,

Cindy Miller,

Research

Foundation,

of Canada.

discussion

regarding

and Sandy Nakashima

of donor blocd saples.

REFERENCES 1. 2. 3. 4.

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

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No.

1, 1985

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RESEARCH

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Ccckcroft, S., Gmperts, B. D. (1985) Nature 314, 534-536. Katada, T., Vi, M. (1982) Proc. Natl. Acad. Sci. USA 79, 3129-3133. Nishizuka, Y. (1984) Natxe 308, 693-698. Tsien, R. Y., Pozzan, T., Rink, T. J. (1982) J. Cell Biol. 94, 325-334. Katada, T., B&och, G. M., Northup, J. K., Ui, M., Gilmn, A. G. (1984) J. Biol. Chem. 259, 3568-3577. T. F. P., Rorgeat, P., Wnite, J. R., Naccache, P. H., Molski, Sha'afi, R. I. (1983) Biochem. Bicphys. Res. Cm. 113, 44-50. Katada, T., Bokoch, G. M., ui, M., Gilman, A. G. (1984) J. Biol. Cl-em. 259, 3578-3585. Bokoch, G. M., Gilman, A. G. (1984) Cell 39, 301-308.

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