Phorbol myristate acetate stimulates ATP dependent calcium transport by the plasma membrane of neutrophils

Phorbol myristate acetate stimulates

ATP-dependent calcium transport by the plasma

membrane of neutrophils.

H Lagast, … , F A Waldvogel, P D Lew

J Clin Invest.

1984;

73(3)

:878-883.

https://doi.org/10.1172/JCI111284

.

We studied the effect of phorbol myristate acetate (PMA) on the plasma membrane

ATP-dependent calcium pump in neutrophils. Plasma membrane-enriched fractions

("podosomes") from PMA-stimulated guinea pig neutrophils exhibited a twofold stimulation

of ATP-dependent calcium transport when compared with control podosomes. The

stimulatory effect was rapid (beginning less than 2 min after exposure to PMA) and reached

maximal values within 5 min. PMA increased the maximum velocity but not the affinity of the

calcium pump for Ca++. Pump activation was not preceded by a rise in cytosolic free

calcium concentration [Ca++]i, as assessed by the intracellularly trapped fluorescent

calcium indicator Quin 2, but instead slightly lowered [Ca++]i and prevented the rise in

[Ca++]i normally induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine.

These results suggest that the calcium pump in the plasma membrane of neutrophils may

be stimulated by calcium-independent pathways, and that this activation could be one of the

earliest events mediating some of the effects of phorbol esters.

Research Article

Rapid Publication

Phorbol Myristate

Acetate

Stimulates

ATP-dependent

Calcium Transport

by the

Plasma Membrane

of

Neutrophils

Hjalmar Lagast, TullioPozzan, Francis A.Waldvogel, and P. Daniel Low

Infectious Diseases Division, Department of Medicine, University Hospital of Geneva, Switzerland, andIstitutodiPatologia Generale, University of Padova, Italy

Abstract. We studied the effect of phorbol

my-ristate

acetate

(PMA) on the plasma membrane

ATP-dependent

calcium

pump in

neutrophils.

Plasma

mem-brane-enriched fractions ("podosomes") from

PMA-stimulated guinea pig neutrophils exhibited

a

twofold

stimulation of ATP-dependent calcium

transport when

compared with control podosomes.

The

stimulatory effect

was

rapid

(beginning

less than 2

min

after

exposure to

PMA) and reached

maximal

values

within

5 min. PMA

increased

the

maximum velocity

but not the

affinity of

the

calcium

pump for

Ca".

Pump

activation

was not

preceded by a

rise

in

cytosolic

free calcium concentration

[Ca++Ji,

as

assessed

by the

intracellularly trapped fluorescent calcium

indicator Quin

2,

but

instead slightly lowered

[Ca+J]i

and

prevented

the

rise

in

[Ca++]i

normally induced

by the chemotactic

pep-tide

formyl-methionyl-leucyl-phenylalanine.

These results suggest that the

calcium

pump in the

plasma membrane

of neutrophils

may be

stimulated by

calcium-independent pathways,

and that this activation

This workwaspresentedinpartattheNationalMeetingof the American

Federation for ClinicalResearch, Washington, DC, 29 April-2 May 1983,and waspublished in abstract form in Clin.Res.1983.31(2):410A.

Dr.Lagast is therecipientofafellowshipfrom the FoundationJean andRoseHoguet,Brussels,Belgium.Hispresentaddressis Service de Medecine Interne, Institut JulesBordet,Brussels, Belgium.Dr. Lew is therecipientofaMaxCloetta CareerDevelopmentAward.

Receivedfor publication26September1983 andinrevisedform8 December 1983.

could be one of the earliest events mediating some of the

effects of phorbol esters.

Introduction

Phorbol esters are potent tumor promoters (1), inducing the expression oftransformation-like properties in a variety of

un-differentiated cell lines in vitro (2, 3). In addition, they are

capable ofstimulating many cells including neutrophils (4-6). Phorbol esters have been shown to bind to specific cellreceptors

in order to exert their functions (7). Several studies indicate

that intracellular calcium may be involved in the effect of phorbol esters (8).Arecentstudy hasshown that uponexposure tophorbol myristate acetate(PMA),' asignificant proportion oftotal neutrophil calcium is extruded into the extracellular medium, suggesting stimulation of calcium extrusion mecha-nisms (9).

We haverecentlydemonstratedthat inside-out vesicles from theplasma membrane of PMA-treated guinea pig andhuman

neutrophils canaccumulate large amountsof calciumin a

cal-modulinsensitive,Mg-ATP-dependentprocess(10). The calcium

accumulationhas been attributedto aplasmamembranecalcium

pump with characteristics similar to that described in other

tissues (11, 12).

Ananalysis of the effect ofPMA on theplasmamembrane

calcium pumping activity and on the levels of free cytosolic calcium

([Ca++]i)

inneutrophilsforms the basis of thisreport.

Methods

Chemicals.Quin2 AMwaspurchasedfrom LancasterSynthesis, England;

lonophoreA23 187fromCalbiochem-Behring Corp.,American Hoechst

1. Abbreviations used in this paper: 4a-phorbol, 4a,9a, 12,B, I 3a,20-pen-tahydroxytiglia-1-6-dien-3-one;

4(#-phorbol,

4#,9a,

12fl,

13a,20-penta-hydroxytiglia-1-6-dien-3-one;[Ca+]j,free cytosolic calcium

concentra-tions; FMLP, formyl-methionyl-leucyl-phenylalanine; PMA, phorbol myristateacetate.

J.Clin. Invest.

©_{TheAmerican Society} for ClinicalInvestigation,Inc. 0021-9738/84/03/0878/06 $1.00

Corp., San Diego, CA; PMA, 4a,9a,12(B,13a,20-pentahydroxytiglia-l-6-dien-3-one (4a-phorbol), 413,9a, 1213,13a,20-pentahydroxytiglia-1-6-dien-3-one (4f3-phorbol), and formyl-methionyl-leucyl-phenylalanine (FMLP) were obtained from Sigma Chemical Co., St. Louis, MO.

ATP-dependentcalciumuptakeby plasma

membrane-enrichedfrac-tionsfrom guinea pig neutrophils. Preparation of plasma membrane-enrichedfractions andmeasurementofATP-dependent calcium transport was carried out as previously described (10).

In brief, neutrophils purified from guinea pigperitonealexudates were incubated for 5 min at370Cin amedium containing 130 mM NaCl, 4 mMKCl,50MMCaC12, and 10mMsodium phosphate buffer, pH 7.4, in the absence or in the presence ofPMA(1.6 MM). Plasma membrane-enriched fractions ("podosomes") were subsequently obtained by gentle sonication and differential centrifugation. Ca uptake by po-dosomes was measured in a medium containing 100mMKCI; 30 mM imidazole-HCI, pH 7; 5 mM azide; 5 mM Mg-ATP; and various con-centrations ofCaCI2 containing 45Ca and EGTA by the Millipore filtration technique(Millipore Corp.,Bedford, MA). Ca uptake rates were expressed as nanomoles Ca per milligram total protein per minute after the values bound in the absence of ATP were subtracted. In the absence of ATP, <O.5 nmol Ca/mg protein became associated with the membranes in <1 minand the valuesdidnotincrease thereafter.

Thefree Caconcentration of solutions in the presence of ATP with orwithout EGTA were calculated by means of the computer program of Perrin and Sayce (10). The calculated values correlated well with direct measurements of free calcium performed with calcium electrodes, as described previously (10). Bovine brain calmodulin used in these experiments was obtainedasdescribed previously (13). The proportion of inside-out vesicles in the membrane preparation was assessed by the activity of 5'-nucleotidase, an ectoenzyme of guinea pig granulocytes, in the presence or absence of Triton X- 100, as described previously (10).

A

z -6 E C E y m u

Measureofcytosolicfreecalciumbythe intracellular

fluorescent

Ca indicatorQuin2.Theisolatedguinea pigneutrophilswereresuspended inamedium containing 138 mMNaCl,6 mMKCI, 1.2 mMPi, 1.2 mMMgS04, 1mMCaC12,5.6 mMglucose,5mMNaHCO3,and 20 mMHepes(pH7.4at37°C), supplementedwith10%autologousserum, until used.Quin2loadingwasperformedasdescribedpreviously(14). Thecalibration ofQuin 2 fluorescenceas afunction ofcytosolicfree Ca++wasdescribedpreviously (14).Excitation and emissionwavelengths

were339±6 and 492±5 nm,respectively.

Results

Fig. I Ashows that whenplasmamembrane vesiclesareisolated fromguineapig neutrophils previously treated for5 min with PMA, therateofATP-dependent Ca accumulation ismorethan doubled compared with vesicles prepared without PMA. Ad-dition of the calcium ionophore A23187 released

45Ca

in the

supernatantfrom both PMA and control vesiclestolevels ob-tained in the absence of Mg-ATP, indicating that calcium had been accumulated byboth vesicle preparations against a

con-centration

gradient.

No difference in 45Ca associated with both membrane

prep-arationswas seen inthe absence of ATP, indicating that PMA doesnotchangecalcium bindingtothe plasma membrane. The

specificity of the stimulatory effect by PMA on the calcium

pumpin theplasmamembranewasstudiedbyincubating

guinea

pigneutrophils withaninactive phorbol analogue,

4f3-phorbol.

4f3-phorbol

(1.6 ,gM) slightly

inhibited active calciumtransport

when compared with vesicles prepared from neutrophils

in-B

3 minutes 15 - 4- I-ci <D 'h

E- 5

-m

0 _-II

0 10 12 14 16 minutes

Figure. 1.(A)Calciumuptake by

plasma-enriched fractions from

guinea pigneutrophils thatwere in-cubated in the presence(-o-)or

in the absence(- ---)of PMA. IonophoreA23187(5MM)

wasaddedat3 min. (B) Effect on calcium fluxesbytheloweringof free Ca++ in the incubation

me-dium.Calcium uptakewas per-formedover 10min,asdescribed in A,by membranes prepared in the sameway( , with PMA;

---withoutPMA).After 10 min of up-take,analiquotwasremovedfrom theincubation medium for 45Ca de-terminationandeither EGTA (A,*)

orthesamevolume of buffer (A, o) wasadded. Ca uptake was also de-terminedat 12, 14, 16,and 20 min of experimentation. Theaddition of EGTAlowered free Ca in the incu-bation medium from pCa4.9 to pCa 7.6.

EGTA --_-°-'0

r<-*---^

----.-Buffer

cubated in the absence ofaphorbolester(25±10% inhibition, mean±SD, n =4).

Twolines of evidence indicate that PMApretreatment

spe-cifically increases the calcium pumping activity rather than stimulatingcalcium accumulation by indirect effects: (a) The proportion of inside-out vesicles,asassessed by5'-nucleotidase

activity, an ectoenzyme of the guinea piggranulocyte, in the

presence orabsence of TritonX-100,is similar forcontrol

ves,-iclesand vesicles from PMA-treated neutrophils, i.e., 45±8%

and 50±5%,respectively (mean±SD, n= 9).(b)Nodifference

in the passive Ca permeability between thetwopopulation of vesicles isobserved:Fig. 1 Bshows that the addition ofEGTA,

when Ca uptake isalmostcompleted, inducesaslow releaseof

45Ca into the supernatant; thekinetics of release is similar in controls and PMA-pretreated vesicles. In both preparations, the

percentageofCareleased in the10min following EGTA addition

is -20%of the uptake during the first 10 min oftheexperiment.

Fig. 2 A shows that the stimulation of Ca accumulation by the vesiclesobtained from PMA-treated neutrophils requiresat

least I or2 min of preincubation ofthe phorbol ester with

intact cells anditis maximal after about5min. The lagphase

ofthis activation is similartothe latency of PMA-induced

su-peroxide production in intact cells (15). Fig. 2 B shows that preincubation with PMA increases the maximum velocity

_(V.)

butnotthe Michalisconstant(Ki) of Catransport.TheKm of

Ca"+

of calciumtransportbyvesicles prepared in thepresence

of PMA was0.38±0.12 gM

Ca",

whereas the

K.

of Ca++ of

those vesicles prepared simultaneously in the absence of this phorbolesterwas0.43±0.04 ,M Ca++ (n=3, mean±SD). This

effect is different from the stimulatory effects of calmodulinon

plasma membrane Ca-ATPases, which affects both the

K.

and the

_Vm.

of Catransport(11). Furthermore, addition of

exog-enouscalmodulin (10

_jig/ml)

to control andPMA-pretreated vesiclescauses nofurtherincreaseofCa accumulation, indicating

that enoughcalmodulinremainsboundtothe Capumpunits.

When theexperimental conditionsarereversed, i.e., when PMA (1.6

_AM)

is addedat37°C for 5 mintoplasma membrane vesicles isolated from untreated cells,nosubsequent activation

ofthe rateofcalcium accumulation is observed. These results

suggestthatPMAactivationrequiressomecomponent(s)present inintact cells, whicharelostduring vesicle preparation. However, asshownbyourresults, the effect ofPMAonCauptake

with-stood the manipulations required to isolate the vesicles and

persisted for several hoursaftertheir isolation.

Assessment of free cytosolic Ca with the intracellularly trapped fluorescent Ca indicator Quin 2 allowed us to study the effect ofPMAaddedtointactguineapigand human

neu-trophils. Upon addition ofPMA (100 nM), cytosolic free Ca

didnotrise, but decreased slightlybelow therestinglevel after alag time <1 min (Fig. 3 C). Fig. 3 C shows also that PMA

prevented almost completely the cytosolicfree Ca rise induced

by thechemotactic peptide, FMLP. This effectwasnotedeven

15minafter the addition of PMA (nottestedthereafter).In the

absence ofPMA,FMLP induced arise ofcytosolicfreeCa to

A

,

q}

0.

E

-R c

x

co

a

4J

100

50

0

0 1 2 3 4 , 5

Incubation time withPM (min)

B

10

5 -Z

ct

E

19

0

Free calcium

(u

M

Figure 2. (A) Timecourseof PMA activation.Neutrophilswere

incu-batedwith PMA(1.6 MM)forvarious durations oftime; plasma

membrane-enrichedfractionswereobtained and tested for initial Ca uptakeat3min after addition of Mg-ATP. At0and5min

incuba-tion,meanand SD(bars)ofsevenexperimentsareshown. (B)Effect

of PMAonthe

_K.

and V..axof Calciumtransport.Asplotted from

thisgraph, membranespreparedwithoutPMA(- - - - --)hada

K.

forCa of 430 nM andVm.of0.55nmolCa/mgproteinpermin,

and themembranespreparedsimultaneously withPMA(1.6gM)

(-0-) exhibiteda for Caof 518 nMandVm0 of 1.05nmol

Ca/mg proteinpermin.

800 nM in the samebatch ofQuin 2 loaded cells(Fig. 3A). Moreover, PMA added after FMLP accelerated the return of

cytosolicfree Ca to basal levels(Fig.3B). Qualitativelysimilar

tracingswereobtained inneutrophilsfromapatientwithchronic

granulomatous disease and in guinea pig peritoneal exudate

neutrophils.At 10nM,PMAadded 3minbefore the addition (7)

[Caelo

=

lo-,

M

50-0

(-in

+u

-6.2 X10-7

[Cati,

M

-1.42X10-7

100- PMA -10-3

FMLP

4.1

-0

100

-

5

0-of FMLP (2 X 10-7 M) significantly increased the return of

cytosolicfree calciumtobasal levels inducedbythechemotactic

peptides (4.5±1.2 min instead of 8±1.5 min for a batch of human neutrophils loaded with 0.6 nmol Quin 2/106 PMN; mean±SDofn = 5,P<0.02by pairedttest). Bycontrast,no

effect of the FMLP-induced changesin free cytosolic calcium

wasobserved when human neutrophilswerepreincubated with two inactive phorbol analogues, 4a-phorbol (100 nM) or

4j3-phorbol (100 nM).

Discussion

Three main mechanismsmayexplain the stimulatory effectof PMA on the ATP-dependent Ca pumping activity present in

[Cat

10-7

X10-7

~+]i

M

[Cae+],

M

-1.6-10-7

1.31 10- Figure3. Effect ofPMA and FMLPonthe levelsofcytosolicfree Ca in human

neutro-phils, loaded withQuin 2. PMA= 100nM, FMLP =0.1 sM.Experiments performed in

the samebatch of cells (Quin 2 loading: 1.2

-10-9 nmol/1I 6 PMN).

theplasmamembrane ofneutrophils: PMA pretreatment may

increasethe number of Capumpunits in theplasma membranes;

PMAcould modify qualitativelytheplasmamembrane Capump

units andtherebyincrease their Vman;and PMA could alter the

membrane lipidstructuressurroundingthepumpunits. As far

asthe first mechanism is concerned, one could postulate that

PMA induces a recruitment ofpreviously inactive Ca pump

unitsin theplasma membrane,ormodifies therecyclingofthe Capump monomersfrom the membranetointracellular vesicles and vice-versa. In support ofthis, PMA is known to induce

exocytosis of secondary granulesin neutrophils (16); although

the presenceofcalcium pumpsinthe primary andsecondary

granuleshasnotbeen demonstratedyet,suchalocation would

leadtoanincrease of the number of Capumpunitspersurface

FMLP

I_

___ ___

PMA

FMLP

$1

area after their fusion with theplasma membrane. Thesecond

possibility is more likely, since it is consistent with themore

general effect of phorbol esters on calcium homeostasis in a number of cell types including lymphocytes, which possess a very limited amount ofintracellular vesicles (17). One likely candidate reaction for an enzymatic modification of neutrophil Ca-ATPase is its direct or indirect phosphorylation by protein

kinasesactivated by PMA. This hypothesis is in agreement with at least three well-documented facts: (a) phosphorylation of phospholamban is known to activate the Ca pump of sarco-plasmic reticulum and sarcolemma by cAMP-dependent kinases (18, 19); (b) phorbol esters are known to activate protein c

kinaseinvitro, and phorbol ester receptors are copurified with protein c kinase (20); and (c) phosphorylation of four major bands are known to be an early event in the interaction of PMA with intact neutrophils (21).

As athird possibility, an effect of PMA on lipid fluidity has been suggested on the basis of its effects on 1 ,6-diphenyl- 1,3,5-hexatriene fluorescence (22). Though the results obtained with

this probe are difficult to interpret in systems as complicated

asintact cells, aneffect ofPMA onlipid domains surrounding the plasma membrane calcium pump cannot be excluded.

Whatever the mechanism of PMA stimulation ofcalcium pumping activity, this activation results inasmall decrease of resting free Ca"+ andinhibition of the

[Ca++Ji

rise induced by chemotacticpeptides.Itisimportanttonotethatsimilarresults

on the effect ofphorbol esters on the levels ofcytosolic free calciumusing Quin2 wereobtainedpreviouslyinlymphocytes

by Tsien et al. (17), indicatingthat theeffect of phorbolesters on calcium pumps isawidespread phenomenum.

Besidesdemonstratingthatoneof theearliestphysiological eventselicitedbyPMA onneutrophilsis the stimulation of Ca

extrusion viatheplasmamembrane Ca pump,ourstudies suggest thatthis occurs byaCa-independent pathwaybecauseit isnot

preceded byarise incytosolic freecalcium, noritismediated by analteration in theaffinity constantofthe pump for Ca.

Recent data would suggest thatdiacylglycerol produced by stimulus-dependentbreakdown of

phosphoinisitide

is involved in thiscalcium-independent activation (23).

Itis generally believed that Ca pump stimulationoccursif cytosolic free calcium is increased because more substrate is availabletothe pump, and

possibly,

because of

secondary

in-creased calmodulin binding (23). However, the stimulation of

the calcium pumpbyincreased

[Ca++]i

isareversible phenom-enonand theactivityreturns tobasal levels when

[Ca++]i

returns torestingvalues. On the contrary, PMAstimulation seemsto

beessentiallyirreversible in intact cells andin isolated vesicles.

Overall, these results suggest that the

plasma

membranecalcium

pump maybeactivated byvariousdifferent mechanisms. The

demonstrated stimulation of the Ca

pumping activity

of the

plasma membraneprovidesa

simple

mechanismto

explain

the

effects of PMA on Ca homeostasis in various cell systems. Whetherthis activation ofCa extrusion is a critical factor in

thestimulation ofcell functions remainstobeestablished.

Acknowledgments

WegratefullyacknowledgeProfessor H. J. Schatzmann for hisvaluable advice, MeirKrygerforreviewing the manuscript, Mrs E. Hugglerfor skillful technicalhelp, andA. Dall'aglio for typing the manuscript.

Thiswork wassupported by grants 3.986-0.82 and 3.896-0-81 from the Swiss NationalScience Foundation and by a Consiglio Nazionale delleRicerche to the Unit for the Study ofthe Physiology ofMitochondria.

References

1. Diamond, L., T. G. O'Brien, and W. M. Baird. 1980. Tumor promotorsand themechanism of tumor promotion. Adv. Cancer Res. 32:1-74.

2. Totterman,T. H., K.Nilsson,and C.Sundstrom. 1980. Phorbol ester-induced differentiation of chronic lymphocyticleukemia cells. Na-ture(Lond.). 288:176-179.

3.Rovera, G., D. Santoli,andC. Damski. 1979. Human promy-elocytic leukemia cells in culturedifferentiate intomacrophage-like cells when treatedwith a phorbol dester.Proc.Nati.Acad.Sci.USA. 76:2779-2783.

4. De Chatelet, L. R.,P. S. Shirley,and R. B. Johnston, Jr. 1976. Effect ofphorbolmyristateacetate on theoxidativemetabolism of human polymorphonuclear leukocytes.Blood.47:545-555.

5. Zucker, M.B., W.Troll, and S. Bellman. 1974. The tumor pro-moterphorbol ester(12-O-Tetradecanoyl-phorbol-B-acetate), a potent

aggregatingagentforbloodplatelets.J. Cell Biol.60:325-336. 6. Abb, J., G. J. Bayliss, and F. Deimhardt. 1979. Lymphocyte activationbythetumor-promotingagent

12-0-Tetradecanoyl-phorbol-13-acetate(TPA). J.Immunol. 122:1639-1646.

7. Shoyab, M., and G. J. Todaro. 1980.Specific high affinitycell membranereceptorsforbiologically active phorbol and ingenolesters. Nature(Lond.). 288:451-456.

8.Smith,B.M.,F. K.Kessler, S. H.Corchman,andR. A.Corchman. 1982. The roleof calcium in macrophage chemotaxistothe phorbol estertumor promoter TPA.Cell Calcium. 3:503-514.

9.Mottola, C., andD. Romeo.1982. Calciummovementand

mem-branepotential changes in theearly phaseofneutrophilactivationby

phorbolmyristateacetate: astudy with ion-selective electrodes.J. Cell Biol. 93:129-134.

10. Lagast, H., P. D. Lew, and F. A. Waldvogel. Adenosine tri-phosphate-dependent calciumpumpin the plasma membrane ofguinea

pig and humanneutrophils.J. Clin.Invest. 73:107-115.

11.Schatzmann,H.J. 1983. The red cell calciumpump. Annu. Rev. Physiol. 45:303-312.

12.Caroni, P., and E.Carafoli. 1981. TheCa2"-pumping ATPase of heart sarcolemma.J. Biol. Chem. 256:3263-3270.

13.Lew,P.D., andT.P.Stossel. 1981. Effect ofcalciumonsuperoxide

production byphagocytic vesicles from rabbitalveolarmacrophages.J. Clin.Invest. 67:1-9.

14.Pozzan,T.,P. D.Lew,C. B.Wollheim,and R. Y.Tsien. 1983. Is anincreaseincytosolicfree calciumasufficient and necessary stimulus

totriggerO2 productionandexocytosisinhumanneutrophils?Science

(Wash. DC).221:1413-1415.

disease: deficient oxidativemetabolism due to a lowaffinityNADPH oxidase. N. Engl.J. Med. 305:1329-1333.

16. Wright, D. G., D. A. Bralove, and J. I.Gallin. 1977.The dif-ferential mobilization ofhumanneutrophilgranules.Effects ofphorbol myristateacetate andlonophore A23187.Am. J. Pathol. 87:273-283.

17.Tsien,R.Y., T. Pozzan, andT.J. Rink. 1982.Tcell mitogenes cause early changesincytoplasmic freeCa2" andmembrane potential inlymphocytes. Nature (Lond.). 295:68-70.

18.Caroni,P., and E.Carafoli. 1981. Regulation ofCa"2-pumping

ATPase of heart sarcolemma by aphosphorilation-dephosphorilation process. J. Biol. Chem. 256:9371-9373.

19. Tada,M., Y. Yamada, M.Kadona, M. Inuri,and F.Ohmori. 1982. Calcium transport by cardiac sarcoplasmic reticulum and phos-phorilation of phospholamban. Mol. Cell. Biochem. 46:73-95.

20. Niedel, J. E., J. L. Kuhn, and G. R. Vanderberk. 1983. Phorbol

diester receptor copurifies with protein kinase C. Proc. Natl. Acad. Sci. USA. 80:36-40.

21. Andrews, P.C.,and B. M. Babior. 1983. Endogenous protein phosphorilation by resting and activated human neutrophils. Blood. 61:333-338.

22.Stocker, R.,K.H.Winterhalter, and C. Richter. 1982.Increased fluorescence polarization of 1-6-diphenyl-l-3-5-hexatriene in the phorbol myristate acetate-stimulatedplasmamembrane ofhumanneutrophils. FEBS(Fed. Eur.Biochem. Soc.) Lett. 144:199-203.

23. Rink,T.J., A.Sanchez, andT.J. Hallam. 1983.Diacylglycerol and phorbol esterstimulate secretion without raising cytoplasmic free calcium in human platelets. Nature (Lond.). 305:317-319.