Phospholipase C and phospholipase D are activated ... - CiteSeerX

4 downloads 0 Views 1MB Size Report
C and phospholipase. D are activated independently of each other in chemotactic peptide-stimulated human neutrophils. Theodore. J. Mullmann,. Boonlert.
Phospholipase

C and phospholipase

independently human

of each

in chemotactic

peptide-stimulated

neutrophils Theodore J. Mullmann, Robert W. Egan, and Schering-Plough

Abstract:

other

D are activated

cytochalasin

Institute,

Cheewatrakoolpong, BiIlah

Kenilworth,

B-treated

New

C. Anthes,

Marvin

I. Siegel,

Jersey

late

most PLC,

[2].

In

containing phosphoglycerides to form choline and diradyl-sn-glycero-3-phosphate (phosphatidic acid), suggesting a possible link between PLC and PLD. However, in the absence of cytochalasin B or extracellular Ca2, PLC was fully activated by fMLP with minimal activa-

potent

tion of PLD, sufficient for

down might be necessary for PLD activation. Because PKC inhibitors such as K252a and staurosporine do not inhibit PLD in fMLP-stimulated neutrophils [9-il], it is likely that diacylglycerol and Ca2 mobilized during fMLP stimulation act directly at the level of PLD and not through PKC. Subse-

indicating that PLD activation.

required

the

PLC Full

simultaneous

neutrophils

John

stimulated with fMet-Leu-Phe (fMLP) in the presence of Ca24, phospholipase C (PLC) activity, as measured by inositol-1,4,5-trisphosphate (1P3) formation, preceded phospholipase D (PLD)-catalyzed breakdown of choline-

fMLP

When

Research

Boonlert M. Motasim

activation activation

were

alone is not of PLD by

presence

of both

Ca24

and cytochalasin B, a condition that caused no further enhancement of PLC. This result suggests that PLD products are not involved in the regulation of PLC activation. tion there

activate PLD in the absence of PLC. Treatment of intact neutrophils with pertussis toxin inhibited both PLC and PLD, with PLC inhibition occurring at lower concentrations than PLD inhibition. These differential effects of pertussis toxin and the observed lack of inhibition of

distinct

G-proteins.

Taken

together,

suggests that, in fMLP-stimulated PLD are activated through Leukoc. Biol. 53: 630-635; Key Words: G-proieins calcium diglycerides

pert

is believed activation, receptors

these

to inimply through

observations

neutrophils, PLC independent mechanisms. 1993. ussis

toxin

signal

are

phorbol

Ca24

ionophore

diacylglycerol

ester

activators

phorbol

A23187,

the

are

with the phospholipase

in many

cells

accumulations

of PLD

(PMA)

including

neutrophils

products,

PA and

rapidly substrates.

Biology

pepand

activated to hydrolyze PLC hydrolyzes (PIP2) to produce 1,2-

Volume

53, June

perand

13-acetate

[2, 6, 9]. More important, 1-oleolyl-2-acetylglycerol PMA act synergistically with A23187 to activate granulocytes [9], raising the possibility that initial in diacylglycerol and Ca2 due to phosphoinositide

are

and PLD in increases break-

DG,

in large

quantities, may, in turn, exert modulatory effects on PLC. This possibility is indicated by the fact that the addition of PA to fibroblasts activates PLC [12, 13] and that PMA, a diacylglycerol mimic, inhibits PLC in many cells including neutrophils [14, 151. The objective of the present study was to examine the relationship between PLC and PLD in fMLP-stimulated neutrophils. By using both kinetic and pharmacologic proaches, we have obtained data suggesting that activated in the absence of PLC, that PLD products involved in PLC activation, and that PLC and PLD pled to fMLP receptors through distinct G-proteins.

MATERIALS

transduction

chemotactic C (PLC)

cell

1-oleolyl-2-acetylglycerol,

12-myristate

of PLD

PLD also stimuCa2 mobilization

apPLD is are not are cou-

AND METHODS

Materials 1-O-[ 3H]Alkyl-2-lyso-sn-glycero-3-phosphocholine mmol) and I-O-alkyl-2-lyso-sn-glycero-3-phospho[3H]choline (79 Ci/mmol) were purchased from Dupont-NEN.

stimulated (fMLP),

of Leukocyte

the

activate and

J.

diacylglycerol and inositol-1,4,5-trisphosphate (1P3) [1], and choline-containing phosphoglycerides (PCs) are hydrolyzed by PLD to produce diradyl-sn-glycero-3-phosphate (phosphatidic acid, PA) and choline [2, 3]. This PA is then metabolized by PA phosphohydrolase to l,2-diradyl-snglycerol (DG) [4-6]. IP3 mobilizes Ca2 from intracellular stores [7], and DG acts in concert with Ca2 to activate protein kinase C (PKC) [8].

Journal

cells, agents that to DG production

addition, synthetic

binding

phospholipase D (PLD) specific phospholipid phosphatidylinositol-4,5-bisphosphate

630

the

other leading

and

INTRODUCTION When neutrophils tide fMet-Leu-Phe

meant

quent

Furthermore, under conditions of complete inhibiof PLC by phorbol 12-myristate 13-acetate (PMA), was no inhibition of PLD, showing that fMLP can

fMLP-stimulated PLD by PMA, which activate G-proteins involved in PLC that PLC and PLD are linked to fMLP

In

1993

lipids

kits were

were purchased

obtained from

from Avanti

(100

1P3

Amersham.

Unlabeled

Polar

Precoated

Lipids.

Ci/

Abbreviations: fMLP, formyl-Met-Leu-Phe; CB, cytochalasin B; PLC, phospholipase C; 1P3, inositol-1,4,5-trisphosphate; PLD, phospholipase D; PC, choline-containing phosphoglycerides; PA, diradyl-sn-glycerol-3-3phosphate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PT, pertussis toxin; DG, diradyiglycerol; alkylacyl-GP, [1-O-alkyl]-2-acyl-snglycero-3-phosphate; alkylacyl-GPC, l-O-alkyl-2-acyl-sn-glycero-3-phosphocholine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EGTA, jethylene-bis(oxyethylenenitrilo)ltetraacetic acid. Reprint requests: TheodoreJ. Mullman, Schering-Plough tute, 2015 Galloping Hill Road, Kenilworth, NJ 07033-0539. Received January 7, 1993; accepted February 5, 1993.

Research

Insti-

silica

gel

Pertussis

G (0.25 toxin

purchased

mm thick) was purchased (PT) was from Calbiochem.

from

Pharmacia-LKB

cytochalasin B (CB), PMA, and all other Chemical Co.

Isolation

Biotechnology.

fatty acid-free reagents were

and labeling

from Whatman. Ficoll-Paque was

of human

bovine purchased

Assays

fMLP,

serum

albumin, from Sigma

the 1-O-alkyl chain or the choline moiety as described previously [10]. Briefly, cell mixed with either 1-O-alkyl-2-lyso-sn-glycero-3(1 tCi/107 cells) or 1-O-alkyl-2-lyso-sn-

glycero-3-phospho[ tion for 45 HEPES-saline

beled

cells

Incubation

of sus-

5H]choline (2 1tCi/107). Following incubaat 37#{176}C, cells were washed twice with buffer to remove unincorporated label. La-

then

resuspended

in the

done

same

buffer.

in

duplicate

was performed representative

or

triplicate.

at least experiments.

twice.

The

Each

set

data

presented

of ex-

AND DISCUSSION

PLC and PLD are activated independently in fMLP-stimulated neutrophils

of each other

To examine the temporal relationship between the activation of PLC and PLD, human neutrophils were labeled in alkylacyl-GPC with 3H in the 1-O-alkyl chain. To measure simultaneously the two products of PLC and PLD, [3H]alkylacyl-GPC-labeled cells were stimulated, cell reac-

tions

mm were

were

periments are from

RESULTS

neutrophils

Neutrophils were obtained from human blood that was collected in the anticoagulant acid-citrate-dextrose as described [16]. Isolated neutrophils were suspended in HEPES-saline buffer containing 125 mM NaC1, 0.7 mM MgC12, 10 mM glucose, 0.5 mM EGTA, 1.0 mg/mI fatty acid-free bovine serum albumin, and 25 mM HEPES, pH 7.4, and labeled with 3H in either alkylacyl-GPC pensions were phosphocholine

Data presentation

were

stopped

with

perchloric

acid,

and

the

resulting

precipitate was separated from the supernatant. The lipidcontaining pellet was analyzed for [3H]alkylacyl-GP (PLD activity) and the supernatant was analyzed for IP3 mass (PIP2-specific PLC activity). Activation of PC-specific PLC has been shown in other systems [2]. However, by labeling

conditions

In some experiments, cells were incubated with PT for 2 h at the indicated concentrations. Assay conditions were essentially as described [4-6]. Briefly, assay mixtures in a final volume of 250 1d contained 1.5 mM CaCl2 and 5 tM CB (unless indicated otherwise). In some experiments, PMA was also added to the assay mixture. To these mixtures were then added prelabeled cells (5 x 106) and the suspensions were incubated for 5 mm (37#{176}C) before initiating the reactions by adding either 100 nM fMLP or buffer. At the appropriate times, the reactions were stopped by either perchloric acid for IP3 measurements or chloroform-methanol (1:2 by volume) for lipid analyses. The final concentration of dimethyl sulfoxide in the assays did not exceed 0.1%, a concentration that had no discernible effects.

Simultaneous

measurement

G) 0 F-

a 1

0

E 1) 0

of PLC and PLD

To measure IP3 mass and 1-O-[3H]alkyl-2-acyl-sn-glycero-3phosphate ([3H]alkylacyl-GP) simultaneously, reaction mixtures containing [3H]alkylacyl-GPC-labeled cells and other appropriate additions were stopped with 50 d of ice-cold 20% perchloric acid. After 20 mm on ice, these centrifuged at 2000g for 30 mm. The resulting were then analyzed for IP3 mass as described lipid-containing extracted with

Cl)

pellet was suspended chloroform-methanol

in 250 (1:2 by

samples

were

E

a-

supernatants

below

and

jd of water volume).

the and

1P3 mass assay

10000

>. 0 15

=

The supernatants of perchloric acid-treated cells were neutralized by the addition of 16 il of 10 N KOH followed by 25 d of 1 M Tris, pH 9.0. The pH of the samples was measured and samples that remained acidic were adjusted to pH 9.0. The precipitates of these neutralized samples were removed by centrifugation for 30 mm at 2000g. Aliquots (100 d) of these supernatants were then analyzed for 1P3 quantification, using 1P3 binding kits.

Assay of PLD products

C,,

TIME, Fig.

1.

Time

stimulated described labeled

by the and

Mullmann

the

cells,

IP,

CaCl2, of buffer (0)

1.5 mM

were

reactions

were quantified raphy, respectively

ci aL

of

neutrophils. in Materials

fore the addition Phases from the lipid-containing pellet were separated procedure of Bligh and Dyer [17]. [3H]Alkylacyl-GP [3H]choline were measured as described [10].

and Neutrophils and Methods.

course

stopped

and or

with

were 5 iM 100

CB nM

perchloric

Phospholipase

described

in

Materials

D/phospholipase

formation

labeled

Samples

by an IP, receptor-binding as

sec

(3H]alkylacyl-GP

in

IMLP-

in 5H]alkylacyl-GPC (250

were

pi)

containing

preincubated

as 5

for

5 mm

x

106 be-

(#{149}). At the indicated times, acid. IP, and (‘Hlalkylacyl-GP kit and by thin-layer chromatog-

IMLP

and

Methods.

C relationship

631

TABLE

1.

Effect

of

Extracellular

Ca2

[5Hlalkylacyl-GP

on

fMLP-Stimulated

IP,

120

and

C

Accumulation.

0

Additions

IP, (pmol/IO’

cells)

‘H]Alkylacyl-GP

(dpm

x

100

10’)

E EGTA EGTA EGTA EGTA

0.8

±

0.2

2.7

±

0.2

+

IMLP

5.0

±

0.4

4.3

±

0.4

+

Ca2

1.0

±

0.3

3.3

±

0

+

Ca2

6.1

±

0.2

12.0

±

0.2

IMLP

+

0 80 C’)

a60

Using

the

samples

and

containing 5 sM no added Ca’

either

fMLP.

GP.

methodology

Reactions

These

were

products

described in the were preincubated

CB or

1.5

stopped

were

mM

Ca2,

after

then

legend

for

10 s for

IP,

and

30 s for

as described

0

1, duplicate 0.5 mM EGTA

5 mm with adding buffer

before

quantified

of Figure

or

100

C

nM

[‘H]alkylacyl-

for

Figure

.2

4#{176}

1. 20

studies lation PLC PLC In

[4] [18],

lack of phosphocholine concluded that PC

in fMLP-stimulated neutrophils. activity was measured. the

presence

produced

of

neither

of fMLP, mum

and by the it has been

1 mM

1P3

and

as

early

rapidly

mass accumudegraded by

only

0

PIP2-specifIc

PMA,

CB, neutrophils Upon addition

5 M

[3H]alkylacyl-GP.

1P3 accumulated 10 s and declining

at

Thus

Ca24

nor

is not

as 5 s, reaching

a maxi-

thereafter

1).

(Fig.

Fig. 3. PMA

inhibition

the methodology described in taining 1.5 mM CaCI,, 5 sM were preincubated for 5 mm After 10 s, the reactions were Figure 1.

The

IP3,

0

reaching

followed

a maximum

PLC-derived Ca2 in PLD On the stimulated Ca2, small PLC

within

diacylglycerol activation.

30-60

Using

the

of

accumulation

s. These

data

confirm

intracellular

IP3

was

produced in

to a near-maximal (Table is not sufficient to

alone

In

experiment,

addition

neutrophils and no

EGTA

level with 1), suggesting

[3H]alkylacyl-GP

activation this

mobilization

fully

of Ca2

activate

in excess

of were added only a that PLD

of EGTA

enhanced [3H]alkylacyl-GP formation sixfold with only minor increases in 1P3 accumulation, further indicating that PLDderived products are inconsequential to PLC modulation. Additional support for lack of PLC modulation by PLD products comes from the observations that wortmannin, protein tyrosine kinase inhibitors, and agents that elevate cellular cyclic AMP levels inhibit PLD [11, 20, 21] while having little or no

E

a. 10000

effect

on

more,

PLC

(ref.

-I

C’) -S

but also by fMLP 0

2

4

6

CYTOCHALASIN Fig. 2. Effect of CB

on

neutrophils.

IP,

and

mass

Using

the

Figure 1, duplicate samples containing concentrations of CB were preincubated buffer (0) or 100 nM fMLP (#{149}). After

I‘Hlalkylacyl-GP,

the

as described

Journal

reactions

in Materials

of

Leukocyte

were

1 2

B, tM

[‘Hlalkylacyl-GP methodology

1 0

8

in IMLP-

formation described

in

the

legend

of

1.5 mM CaCl, and the indicated for 5 mm before the addition of 10 s for IP, mass and 30 s for stopped

and

Biology

and

these

products

were

Volume

53, June

1993

and

of

of PLC. observation of PLC,

our

unpublished

neutrophils

with

These that needed

CB (Fig. 2).Thus, in Ca2-containing

when neutrophils buffer in the

like

PMA

fMLP,

was

added

as

protein

activation Ca2

kinase

are

IMLP-induced the presence

maximal with a formation. CB augmented a dose-dependent manner accumulation (see also ref. that the effects of CB and are not attributable to potent activator of PLD in

ogenously

data). PA

conclusions

production [3H]alkylacyl-GP generation in effects on IP3 ther indicate PLD activation PMA is a

such

Methods.

19

premncubation

the activation rated by the PLD, but not

4

stimulated

and

other hand, when CB-treated in the presence of 0.5 mM

increase

activity.

632

neutrophils.

the legend of Figure 1, duplicate samples conCB, and the indicated concentrations of PMA before the addition of buffer or 100 nM fMLP. stopped and analyzed for IP, as described for

the previously reported conclusion that PLC precedes PLD in fMLP-stimulated cells [19]. This temporal relationship between PLC and PLD and the fact that the combined addition of the membrane-permeable diacylglycerol 1-oleoyl-2-acetylglycerol and the Ca2 ionophore A23187 to neutrophils activates PLD [9] are consistent with a role for

0

quantified

nM

fMLP-stimulated

of [3H]alkylacyl-GP

formation U)

>. 0 15

of IP, mass in

CB inhibitors

nor

of [9].

Further-

effect on further corroboactivation of of not only Ca24 had

were absence

no

stimulated of CB, 1P3

small

increase in [3H]alkylacyl-GP while having minimal 22). These data furextracellular Ca24 on PLC. neutrophils [9]. Un-

PLD requires neither exMore important, agents and PT exert differential

30000

pretreatment (Fig.

did

4),

enhance

fMLP’s

that PMA of its action of PLD

alkylacyl-GP)

ability

to activate

and products are incapable

PLD

(DG and of replacing

CB. These data support the view that PMA and fMLP utilize two distinct PLD activation mechanisms which can operate in parallel and that there is little crosstalk betwen these mechanisms. Thus, the observation that the fMLPstimulated mechanism of PLD activation remains fully func-

20000

E

not

indicating

tional mediated

10000

in

the absence mobilization

of PLC would of DG and Ca24

activation. suggested

Similar in other

-J

epidermal detectable

growth PLC

0

bryonic

C., I

neomycin, an aminoglycoside antibiotic that inhibits PLC by complexing with phosphoinositides, inhibited PLC completely while having only a minor effect on PLD [24]. However, incubation of neutrophils with neomycin (10 mM) for 2-4 h had no effect on fMLP-induced 1P3 accumulation or choline formation (our unpublished observation), presumably because neomycin did not permeate through the neutrophil plasma membrane. Although IP3-induced mobilization of Ca24 from intracellular pools appears to be unrelated to fMLP-induced PLD activation (Fig. 4), this PLD activation mechanism does require the simultaneous presence of both extracellular Ca2 and CB (Table 1 and Fig. 2). PLD activation by Ca24 and nonhydrolyzable GTP analogues in cell-free systems [25] is not affected by CB (our unpublished observation), suggesting that CB does not modify PLD’s interaction with Ca24 or G-proteins. It may, therefore, be hypothesized that, in intact cells, CB acts by facilitating the availability of extracellular

‘U

z = 15000

C,)

10000

5000

0

50

sec

TIME, Fig.

4.

Effect

of PMA

preincubation

on

the

I ‘H]cholmne

in neutrophils. In order to measure product that is metabolically much more

PLD the

cells

instead

were of

1.5 mM

CaCI2, for

dicated

times. (0),

5 tM

5 mm Buffer

()

addition;

with

preincubation

Duplicate

Aliquots

the

control;

PMA

samples

either

addition

from

preincubation with

and

CB,

before

IMLP-induced (‘H]choline, stable than

formation

of

the water-soluble l’Hlalkylacyl-GP,

1-O-a1kyl-2-lyso-glycero-3-phospho’HJcholine

[‘Ha1kylacyl-GPC.

cubated line.

labeled

150

100

buffer

the aqueous (#{149}) preincubation with

followed

or

of buffer

100 or

100

nM nM

5 x PMA

106

were

fMLP

for

factor activity

kidney

Ca24

containing

dissociation systems.

indicate that PLCis irrelevant to PLD

cells

to cellular

of PLD instance,

For

from PLC in Swiss

has 3T3

been cells,

activated PLD in the absence [23]. Furthermore, in human

transfected

with

muscarinic

of any em-

receptors,

PLD.

cells, preinthe

in-

phase were counted for [‘H]chowith buffer followed by fMLP

PMA followed by buffer by IMLP addition.

addition;

(U)

80 effects on PLD activation by PMA or fMLP instance, PT and protein tyrosine kinase PLD activation by fMLP but not PMA, kinase C inhibitors act in a reverse manner. tions demonstrate that PMA activates PLD

[2, 9-11, 20]. For inhibitors inhibit whereas protein These observaby a mechanism

that is distinct from that utilized by fMLP. This knowledge and the fact that PMA is a potent inhibitor of fMLP-induced PLC activation [14, 15] provide the basis for the use of PMA as a tool to examine further the involvement of PLC in PLD activation. Under our assay conditions, preincubation with PMA for 5 mm inhibited fMLP-stimulated PLC activation (as measured by 1P3 accumulation) in a dose-dependent manner with complete inhibition occurring at 100 nM PMA (Fig. 3). Alkylacyl-sn-glycero-3-phospho[3H]choline-labeled cells incubated with PMA and CB for 5 mm produced [3H]choline (Fig. 4), a result consistent with our pervious findings [10]. When neutrophils were incubated with 100 nM PMA for 5 mm and subsequently stimulated with 100 nM fMLP for various greater due

times, the levels than those observed to

of [3H]choline with PMA

produced alone. This

were increase

fMLP

was approximately equal to that produced by fMLP in the absence of PMA exposure. Of note, the rate of fMLP-stimulated choline formation remained unaffected by PMA treatment. Furthermore, in the absence of CB, PMA

Mullmann

z 0 I-

60

I

z 40

20 0.13

0.50

0.25

TOXIN,

PERTUSSIS Fig.

5.

Effect

stimulated of Figure 2 h with buffer

of

on

IP, Using

and

13H]alkylacyl-GP

the

methodology

1, [3H]alkyl-GPC-labeled the and

indicated quadruplicate

and 5 M CB 100 nM IMLP. reactions

were

Materials

and

ci al.

PT

neutrophils.

amounts

human of PT.

samples

were preincubated After 10 s for 1P3 stopped and these

1.00

Cells

pg/mI formation

neutrophils were

containing

washed 5 x

106

twice

were

D/phospholipase

legend

cells,

1.5

quantified

C relationship

as

for

PTfree

with

for 5 mm before the addition (#{149}) and 30 s for [‘Hlalkylacyl-GP products

IMLP-

in the

were incubated

Methods.

Phospholipase

in

as described

mM

Ca2,

of buffer

or

(U),

the

described

in

TABLE

2.

Effect

of PT

on

the

fMLP-lnduced

Formation

of I’HlCholine

in Neutrophils

[‘HiCholine,

dpm

x

Preincubated

with

10’

Total Additions

-

None

PMA IMLP PMA

+

IMLP

“Using the methodology described or absence (-) of PT (1 ag/ml) for cubated for 5 mm before the addition counted for [‘H]choline.

PLC and PLD might G-proteins PT the

inhibits the receptor/G-protein

for 2 h.

PT

PLC and PT treatment

0.3

3.9

±

0.2

-

0.2

12.1

±

0.1

-

-

9.6

±

6.1

±

0.1

5.6

2.2

17.7

±

0.5 0.1

14.4

±

0.1

5.3

2.0

Figure

3, cells

labeled

of buffer

in l-O-alkyl-2-acyl-sn-glycero-3-phospho[’H]choline 1.5 mM CaCI,, 5.0 jaM CB, and fMLP. At the end of 30 s, the reaction was stopped

samples

or 100

nM

through

distinct

not

inhibit

caused activities

dependently enzymes proteins.

634

of each other might be linked The data further

Journal

of Leukocyte

data and

PLC is further subpreparations. For inGTP analogues to and not PLD [28],

indicate PLD

that, are

in fMLPactivated in-

and raise the possibility that to receptors through separate suggest that the availability

Biology

1. Rhee,

Volume

53, June

these 0of ex-

1993

S.G.,

inositol

Suh

P.-G.,

Ryu,

S-H.,

phospholipid-specific

that

this

Lee,

S.Y. (1989)

phospholipase

C.

Studies

of 244,

Science

546-550. 2. Billah,

MM.,

functions

Anthes,

of

J.C.

(1990)

The

phosphatidylcholine

regulation

hydrolysis.

and

cellular

Biochein.

269,

j

281-291.

J.

3. Exton, down.

j

4. Billah, MI. the

(1990) Signaling Biol. Che-m. 265,

through 1-4.

phosphatidycholine

MM., Eckel, S., Mullmann, T.J., (1989) Phosphatidylcholine hydrolysis

phosphatidate

and

stimulated

TJ.,

C5a

activation

major

route

to the

Immunol. 144, 6. Mullmann, T.J., PMA activation

levels

Biol.

production

R.W.,

by PLD

Siegel,

determines

in chemotactic

peptide

264, 17069-17077. Egan, R.W., Billah, M.M. (1990) D in human neutrophils: a Chem.

Egan,

Inositol

and

diglycerides.

R.W., Billah, MM. D in human neutrophils

of phosphatides 170, 1197-1202.

(1987) second

break-

Egan,

of phosphatides

1901-1908. Siegel, MI., of phospholipase

production Res. Commun.

7. Berridge, M.J. two interacting

j

Siegel, MI., of phospholipase

J.

to the ophys.

diglygeride

neutrophils.

5. Mullmann,

PMA-induced

suggesting that PLC and relevant 0-proteins reside in a membrane compartment. By contrast, PLD activation by nonhydrolyzable GTP analogues may require the presence of a factor [29, 30] that is likely to be a 0-protein. Additional reconstitution studies with purified 0-proteins and PLD are needed to demonstrate that the 0-proteins for PLC and present PLC

and

REFERENCES a

fMLP was reduced by 60%, a value identical to that observed for control cells stimulated with IMLP alone (Table 2). These results demonstrate that PMA does not modify PT sensitivity of 0-proteins involved in PLD activation. The notion of unique 0-proteins for PLD and stantiated by experiments with cell-free stance, addition of nonhydrolyzable neutrophil membranes activates PLC

incubated in the presence (+) or PMA (100 nM) were preinfrom the aqueous phase were

tracellular Ca2 to PLD is critical for activation Ca24 availability may be facilitated by CB.

by uncoupling uncoupling occurs [26]. PT and PLD

-

were buffer aliquots

either and

containing

[3H]choline formation (Table 2). However, PMA is believed to inhibit PLC by inactivating PLC-associated G-proteins. Therefore, lack of inhibition of fMLP-induced PLD by PMA (Fig. 4) would indicate that PLC-linked G-proteins might not be involved in PLD activation. Other possible interpretations exist for these results, including the one that PMA may regulate signaling events distal to G-proteins. In order to determine whether PMA modifies the PT effect on PLD, neutrophils were pretreated with PT (1 jzg/ml for 2 h) and then stimulated with PMA (100 nM for 5 mm), followed by stimulation with fMLP for an additional 30 s. Under this treatment protocol, the increase in PLD activity due to

PLD are distinct. In conclusion, our stimulated neutrophils,

PT

+

±

Quadruplicate

did

PT

-

±

of G-proteins of both PLC

PLD. of neutrophils

du e to fMLP

4.0

in fMLP-stimulated cells (Fig. 5). PLC inhibition occurred more readily than PLD inhibition. Likewise, rabbit neutrophils pretreated with PT for varying times displayed dissimilar inhibition curves for PLC and PLD [27]. These results suggest that PT-sensitive G-proteins are involved in the activation of both PLC and PLD and that G-proteins mediating PLC and PLD activities might be distinct. Although the suggestion of two distinct G-protemns for PLC and PLD is a tempting one, alternative explanations exist. For instance, the same 0-protein may have different affinities for

PT

+

of phospholipases interaction. This

through ADP-ribosylation dose-dependent inhibition

Increase

12.4

be activated

activation

PMA

and

diglyerides.

triphosphate Annu.

messengers.

(1990) leads

Biochem.

Bi-

and diacylglycerol: Rev. Biochem.

56,

159-183. 8. Nishizuka, C. Science 9. Billah, MI. cytes:

10.

Y. (1986) Studies 233, 305-309.

ionophore Biol. Chem. Mullmann,

via protein kinase 264, 9069-9076. T.J., Siegel, MI., inhibits

trophils

by

TM.

(1990) three

a protein

kinase

Activation

separable

C

of protein

Egan, R.W., Siegel, D in HL-60 granulodiglyceride and calcium

independent

Egan,

mechanisms.

R.W.,

Billah,

mechanisms.

mechanism.

Zimmerman,

of human

GA.,

neutrophil FASEBJ

j

MM.

phosphohydrolase

C-independent SM.,

kinase

T.J.,

phosphatidate

Chem. 266, 2013-2016. Reinhold, S.L., Prescott, by

perspectives

MM., Pai, J. -K. Mullmann, (1989) Regulation of phospholipase activation by phorbol esters,

Sphingosine

11.

and

(1991) in

neu-

j

Biol.

McIntyre,

phospholipase 4,

D

208-214.

12. Moolenaar, W.H., Kruijer, W., Tilly, B.C., Verlaan, I., Bierman Aj., de Laat, S.W. (1986) Growth factor-like action of phosphatidic acid. Nature 323, 171-173. 13. Knauss, T.C., Jaffer, EE., Abbound, HE. (1990). Phosphatidic acid modulates DNA synthesis, phospholipase C, and platelet derived growth factor mRNAs in cultured mesangial cells. J. Biol. Chem. 265, 14457-14463. 14.

Smith, regulatory

CD.,

human

polymorphonuclear

esters.

15. Tyagi, (1988)

Uhing, protein-mediated

R.J.,

Snyderman, activation leukocytes

BioL Chain. 262, 6121-6127. SR., Tamura, M., Burnham, Phorbol myristate acetate augments

R. (1987) Nucleotide of phospholipase C is disrupted

by

in

phorbol

j

D.N., Lambeth, chemoattractant

J.D. in-

duced

diglyceride

generation

phosphoinositide

16. Boyum, 17.

cytes Bligh,

37, 18.

A. (1968)

from

Biol.

neutrophils

of

mononuclear

Scand. (1959)

j Clin. Isolation

human

blood.

but

263,

Chem.

tween

cells

and

granulo-

Lab. of

Invest. 21, mononuclear

Can. j

Biochem.

Murray, J.J. (1989) and diacylglycerol

R., turnover

polymorphonuclear

leukocytes.

Stimulation production

R.W.,

Tateson,

J.E.,

Garland,

L.G.

(1990)

activation,

production

in

271, 209-213. Uings, I.J., Thompson, Bonser, R.W., Hudson, phosphorylation

R.W.,

Temporal

Spacey,

the N.T., AT., in

human Randall, Garland,

R.W., L.G.

receptor

to phospholipase 597-600.

beand

to

human

A.P.,

diacylglycerol tractants.

III,

M.W.,

stimulates

production Proc. Nail. Acad.

E.G.,

Bi-

Peralta,

J.C.,

Wurtmann,

muscarinic D. j Eckel,

Chain. Siegel,

Gilman, AG. coupling.

(1991)

receptor

266,

factor

Coupling

subtypes

to

6031-6034.

MI.,

Egan,

R.W.,

Phospholipase D in homogenates from implications of calcium and G-protein Biophys. R#{128}s. Commun. 163, 657-664. Casey, P.J., receptor-effector

growth

R.J.

acetylcholine

Biol. S.,

Epidermal

in the absence of inositol Biochein. Soc. Trans. 19, 108S.

(1988)

G-protein

Billah,

M.M.

HL-60 control.

granuloBiochem.

involvement

in

j

Biol. Chain. 263, 2577-2580. 27. Kanaoh, Y., Kanoh, H., Nozawa, Y. (1991) Activation of PLD in rabbit neutrophils by fMLP-mediated pertussis toxin sensitive GTP-binding protein that may be distinct from phospholipase C regulating protein. FEBS LdU. 279, 249-252. 28. Anthes, J.C., Billah, MM., Cali, A., Egan, R.W., Siegel, MI. (1987) Chemotactic peptide, regulation of phospholipase

calcium and guanine nucleotide C activity in membranes from

DMSO

cells.

differentiated

HL-60

145, 825-833. 29. Anthes, J.C., Wang,

Biochem.

Biophys.

Rcs.

Corn-

mun.

Verghese, influx

phospholi-

neutrophil.

Tyagi, SR., Olson, S.C., Burnham, D.N., Lambeth, Cyclic AMP-elevating agents block chemoattractant of diradylglycerol generation by inhibiting phospholipase tivation. j Biol. Chem. 266, 3498-3504. Calcium

j

Spacey, GD., (1992) Tyrosine

coupling

C in the

GD.,

Biochcm.

J.,

transfected

Anthes,

(1991) activity cells.

(1989) cytes:

264,

formation

neutrophil.

M.J.O. D 3T3

phospholipase

relationship

diradylglycerol

is involved

pase D but not ochem. J. 281,

Randall,

Wakelam,

phospholipase hydrolysis in Swiss

Sandmann, of

26.

NT.,

phospholipase

22. Truett,

24.

of in

j

S.J.,

lipid

77-89. cells Physiol.

Biochein.

Cook.

stimulates

25. Snyderman,

superoxide

(1988)

23.

inhibits

13191-13198.

911-917.

Truett, A.P., phosphorylcholine

Bonser,

21.

blood. W.J.

granulocytes

human 909-913. 19. Thompson,

20.

Isolation

from human E.G., Dyer,

and

in human

J.

hydrolysis.

Dillon, a second

in leukocytes Sci. USA.

J.D. (1991) activation D ac-

SB.,

Synderman,

pathway

for

sustained

by

chemoat-

activated 85, 1549-1553.

R.

Mullrnann

(1991)

Granulocyte

P., Siegel,

nucleotide dependent 236-243. 30. Olson, S.C., Bowman, D activation in a cell phorbol 12(3-thiotriphosphate)

ci aL

Phospholipase

MI.,

phospholipase

myristate j

protein

D

factor.

Egan, R.W, Billah, MM. is activated by a guanine Arch. Biochern. PAys. 175,

E.P., Lambeth, J.D. (1991) Phospholipase free system from human neutrophils 13-acetate

BioL

Chain.

D/phospholipase

and

266,

guanosine

by 5-0-

17236-17242.

C relationship

635