Activation of inositol phospholipid breakdown in HL60 cells by P2 ...

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Pa-purinergic Receptors for Extracellular ATP ... of inositol trisphosphate (Imp3) accumulation in these ... lipid breakdown by Pn-purinergic receptors in HL60.
THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 263,No. 34, Issue of December 5, pp. 18108-18117,1988 Printed in U.S.A.

0 1988by The American Society for Biochemistry and Molecular Biology, Inc

Activation of Inositol Phospholipid BreakdownHL60 in Cells by Pa-purinergic Receptors for Extracellular ATP EVIDENCE FOR MEDIATION BY BOTHPERTUSSISTOXIN-SENSITIVE INSENSITIVE MECHANISMS*

AND PERTUSSISTOXIN-

(Received for publication, January 26, 1988)

The mechanisms whereby P2-purinergic receptors types of GTP-binding proteins coupled to phospholifor extracellularATP are coupled to theinositol phos- pase C. pholipid-signaling system were studied in the HL60 human promyelocytic leukemia cell line. Brief pretreatment of either undifferentiated or differentiated HL60 cells with various activatorsof protein kinaseC In another study’ we have demonstrated that extracellular Ca2+/phospholipid-dependent enzyme (e.g. phorbol my- ATP, at nanomolar/micromolar concentrations, triggers rapid ristate acetate) produced a SO-fold decrease in the increases in the cytosolic [Ca”] measured in human phagopotency of extracellular ATP to induce mobilization of cytic leukocytes (neutrophils and monocytes) and in a broad intracellular Ca2+. The ATP-induced increase in rate range of neutrophil/monocyte progenitor cell types including of inositol trisphosphate (Imp3)accumulation in these the HL60 promyelocytic leukemia cell line. Significantly, 4-8-phorboll2-myristate-13-acetate-treated cells was these effects of extracellular ATP on cytosolic [Ca”] were characterized by a 40% decrease in the maximal rate observed in both undifferentiated HL60 cells and in HL60 of InsPs accumulation. Incubation of the cells with NaF cells induced to differentiate along the neutrophil pathway. also induced mobilization of the same Caz+ stores re- In HL60 cells and in allother myeloidcell types tested, leased in response to extracellularATP; this provided extracellular ATP increased cytosolic ICs"] by both mobilizindirect evidence that the transmembrane signaling ing intracellular stores and by activating influx across the actions of Pz-purinergic receptorsmay bemediated by plasma membrane. These actions of ATP are presumably GTP-binding regulatory proteins. This latter possibilproduced in response to occupation of a particular receptor ity wasfurther supported by the findingthat treatment subtype of the so-called P2-purinergic class of cell surface of either undifferentiatedor differentiated HL60 cells receptors (reviewed in Ref. 1).Moreover, the ATP-induced with pertussistoxin produced a significant, but partial, inhibition of ATP-induced signaling actions. These in- Ca2+transients in neutrophils, monocytes, and differentiated cluded 1) a 60-709’0 decrease in themaximum rate of HL60 cells were very similar to those elicited by the chemoInsPs accumulation, and 2) a 1.5 log unit increase in tactic peptide, met-Leu-Phe? This finding raised the possithe half-maximally effective [ATP] required for mo- bility that similar molecular mechanisms might mediate bilization of intracellular Ca2+. In cells treated with transmembrane signaling by both fMet-Leu-Phe receptors both pertussis toxin and4-/3-phorboll2-myristate-13- andthe putative P2-purinergic receptors for extracellular acetate, there was an 80% decrease inmaximal rate of ATP. While the general molecular mechanisms underlying the ATP-induced InsPa accumulation and near-complete inhibition of ATP-induced Ca2+ mobilization. Signifi- coupling between receptors and the polyphosphoinositidecantly, theresidual, pertussis toxin-insensitive portion specific phospholipase C (PI-PLC) have been only partially of ATP-induced signaling was observed in the same defined, both protein kinase C Ca2+/phospholipid-dependent samples of differentiated HL60 cells wherein pertussis enzyme ( 2 , 3 ) and GTP-binding proteins(4-8) appear to play toxin treatment produced complete abolition of InsP3 critical regulatory/modulatory roles. The possible roles of accumulation and Ca2+mobilization in response to oc- these lattersignaling elements in the function of P2-purinergic cupation of chemotactic peptide receptors. These re- receptors have also been investigated in several cell types. sults indicate that the activation of inositol phospho- Charest et al. (9) noted that pretreatment of rat hepatocytes lipid breakdown by Pn-purinergic receptors inHL60 with phorbol esters substantiallyinhibited ATP-induced Ca2+ cells may be mediated by both pertussis toxin-sensitive mobilization. Similar results were observed in Ehrlich tumor and toxin-insensitive mechanisms; this suggests that cells by Weiner et aZ. (10). Irving and Exton (11)have prethese myeloid progenitor cells may express two distinct sented data indicating that, in rat hepatocytes, Pz-purinergic receptors canactivatenot only the polyphosphoinositide-

* This work was supported in part by Grant GM36387 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. 5 To whom correspondence and reprint requests should be addressed Dept. of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, OH 44106. 11 Recipient of a Medical Scientist Training Grant Award.

D. S. Cowen, H. M. Lazarus, S. B. Shurin, S. E. Stoll, and G. R. Dubyak, submitted for publication. The abbreviations used are: fMet-Leu-Phe, formyl methionyl leucyl phenylalanine; InsP’, inositol bisphosphatq Imps, inositol trisphosphate; PMA, 4-0-phorbol 12-myristate 13-acetate; PI-PLC, phosphatidyl inositol-phospholipase C; HEPES, 4-(2-hydroxyethyl)1-piperazineethanesulfonicacid; G-protein, guanine nucleotide regulatory protein.



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specific phospholipase C, but also a phospholipase which 7.5% COz. Under these conditions, the cell doubling time is approximately 2 days (33). HL60 cultures were induced to differentiate into utilizes phosphatidylcholine as a preferred substrate.Preneutrophil-like cells by incubation in the presence of 0.5mM dibutyryl treatment of the cells with pertussis toxin had no marked cyclic AMP for 36-48 h prior to experiments (34). Where indicated, inhibitory effect on the ability of ATP to activateeither HL60 cultures were supplemented with pertussis toxin or cholera phospholipase. The data of Okajima et al. (12) suggested that toxin (List Biologicals) added from sterile 1000-fold stock concenPz-purinergic effects in hepatocytes may be mediated by two trates. Isotopic Labeling of Cellular Phosphoinositides-HL60 cells were ATP receptor subtypes which are coupled to distinct GTPbinding proteins. The first subtype was similar to that char- removed from growth medium by centrifugation andthen resusat lo6cells/ml in serum-free and inositol-free Iscove's medium acterized by Charest et al. (9) and Irving and Exton (11)in pended supplemented with insulin, transferrin, sodium selenite, and 1 pCi/ that itactivated accumulation of inositol trisphosphate via a ml of [1,2-3H]~-myo-inositol (Du Pont-New England Nuclear). The pertussis toxin-insensitivepathway. Occupation of the second cultures were then incubated for 48 h prior to usage. Analysis of lipid receptor subtype with ATP or nonhydrolyzable ATP analogs, labeling by previously described (35) methods showed incorporation but not adenosine, produced inhibition of forskolin-stimu- of 3Honly into thephosphoinositides with average activities of 30,000, lated adenylate cyclase; this effect was blocked in cells pre- 2200, and 1,400 cpm/106cells for phosphatidyl inositol, phosphatidyl treated with pertussis toxin. These data indicate that ATP inositol 4-monophosphate, and phosphatidyl inositol 4,5-bisphosrespectively. receptors may variously be coupled to either pertussis toxin- phate, Analysis of Inositol Phosphates-Cells labeled with [3H]inositol sensitive or pertussis toxin-insensitiveG-proteins. were washed twice with ice-cold basal salt solution containing 125 The HL60 cell line (13-14) provides a unique cell system mM NaC1, 5 mM KC1, 1 mMMgC12, 1.5 mM CaCl2, 25 mM HEPES to further characterize the role of pertussis-toxin G-proteins (Na), 5 mM D-glucose, 1 mg/ml bovine serum albumin, pH 7.4. The in mediating transmembrane signaling by Pz-purinergic cells were resuspended to 1-3 X 106/ml and 0.5-ml aliquots were receptors for extracellular ATP. The activation of phospho- preincubated for 10 min at 37 "C. Agents to be tested were added as lipase C by chemotactic peptide receptors in human phago- 1-5-pl aliquots from appropriately concentrated stock solutions. It should be stressed that all experiments described in this report were cytic cells is probably the best characterized of the pertussis performed in the absence of LiCl as an inhibitor of the terminal toxin-sensitive systems. The inhibitory effects of protein ki- inositol monophosphatase. Reactions were terminated at desired nase C on this response in HL60 cells (15, 16) are similar to times by addition of 0.1 ml of 3.3 N perchloric acid and immediate those observed inhuman (17) or rabbit (18) neutrophils. cooling to 0 "C. After 15-20 min, the acidified extract was centrifuged Similarly, the role of a pertussis toxin-sensitive GTP-binding (8000 X g for 4 min) and 0.5 ml of the supernatantwas removed.The proteinactivated by these receptors has been extensively protein pellet and residual 0.1 ml supernatant were washed with 0.5 ml of 0.55 N perchloric acid and recentrifuged; 0.5 ml of this second studied inboth differentiated HL60 cells (15, 19) and in supernatant was pooled with the first. The combined 1.0 ml of acidic plasma membranes isolated from these cells (16, 20, 21); the supernatant was neutralized by addition of 55 pl of 10 N KOH and functional characteristics of this G-protein appear identical incubation on ice for 15-30 min. After removal of the precipitated to those described in studies utilizing neutrophils or neutro- KCLO4 by centrifugation, 1.0 ml of the neutralized supernatant was phil membranes (6, 7, 17, 22-26). Furthermore, a novel, het- diluted to 10 ml with 5 mM sodium tetraborate, pH 8.5. The diluted erotrimeric GTP-binding protein, which includes a 40-kDa extract was run through a 0.8-ml column of AG1 X 8 resin (Bio-Rad) substrate for ribosylation by pertussis toxin, has recently been in the formate form, and the column was then washed with 24 ml of 50 mM sodium formate, 5 mM sodium tetraborate. Three fractions, purified from both undifferentiated (27) and differentiated respectively containing mono-, bis-, and tris-tetraphosphate esters of HL60 cells (28). The a-subunit of this G-protein, which has myoinositol (36), were collected from each column by successive alternatively been named GHL (28) or Gc (27), can be immu- elution with 12 ml each (collected into 20 ml scintillation vials) of: 1) nologically distinguished from the a-subunits of either Gi or 0.2 M ammonium formate, 0.1 M formic acid, 2) 0.4 M ammonium Go (29). Finally, based on its ability to act as substrate for formate, 0.1 M formic acid, and 3) 1 M ammonium formate, 0.1 M both pertussis toxin and cholera toxin, this HL6O-derived formic acid; between collection of fractions 2 and 3, the column was protein appears identical to a G-protein expressed by neutro- rinsed with an additional 8-ml aliquot (which was discarded) of 0.4 M ammonium formate, 0.1 M formic acid. Each 12-ml fraction was philsand monocytes (30). It appears likely that GHL/Gc then evaporated to dryness and substantially desalted (via sublimafunctionally couples chemotactic receptors to the PI-PLC tion of ammonium formate) by vacuum centrifugation (Savant Speedeffector system. Since previous investigations have used the Vac 200) for at least 18 h. The dried, desalted residues of fractions 1 signaling cascade triggered by such receptors as a hallmark or 2 were dissolved in 1ml of HzOand counted in 10 ml of scintillation for comparison with the biochemical responses triggered by fluid (Formula 963, Du Pont-New England Nuclear); the residue of receptors for other chemoattractants such as platelet-activat- fraction 3 was dissolved in 4 mlof HzO and counted in 14 mlof fluid. ing factor (23, 31), leukotriene B4 (23, 32), and complement scintillation Measurement of Cytosolic[Ca'+]-Undifferentiated or differenfactor C5a (24) we have applied a similar approach to the tiated HL60 cells were removed from growth medium, washed, and characterization of transmembrane signaling by extracellular resuspended at 106/mlin basal salt solution containing 5 mM glucose ATP in both undifferentiated and differentiated HL60 cells. and 1 mg/ml bovine serum albumin. Cell suspensions were preincuThese studies demonstrated that, in both undifferentiated bated for 10 min a t 37 "C prior to addition of 500 nM fura2-AM ester and differentiated HL60 cells, the functional coupling be- (Molecular Probes) from a 1-mM stock solution dissolved in dimethyl tween Pz-purinergic receptors and PI-PLC can be substan- sulfoxide. The suspension was incubated an additional 30-40 min a t 37 "C followed by centrifugation and removal of the supernatant. The tially, but not completely, inhibited by treatment of the cells cells were then resuspended in fresh medium and incubated an with pertussis toxin. This partial inhibition suggests that the additional 10 min at 37 "C. The suspension was recentrifuged, the ATP receptors may interact notonly with the toxin-sensitive cell pellet was resuspended at lo6 cells/ml in ice-cold basal salt GHL/GCprotein, but also with a toxin-insensitive regulatory solution, and the suspension was then stored on ice for up to 4 h during measurements. For such measurements, a 0.3-ml aliquot of G-protein. MATERIALS ANDMETHODS

Cell Preparations-HL6O cells (obtained from the American Type Culture Collection) were routinely cultured under serum-free conditions in Iscove's medium supplemented with 25 mM HEPES, 5 yg/ ml transferrin, and 5 ng/ml sodium selenite. The cells were maintained at 106-106 cells/ml in a humidified atmosphere of 92.5% air,

fura2-loaded cell suspension was diluted into 1.2 mlof basal salt solution (final [cell] = 2 X 106/ml), maintained at 37 'C in a stirred quartz cuvette. Fura2 fluorescence (339 nm excitation/500 nm emission) was measured using previously described instrumentation (35). Agents to be tested were added from 300- to 1000-fold stock concentrates after the basal fluorescence was steady for a t least 4min. Ca2+dependent fura2 fluorescence was calibrated using standard techniques (35,37) after lysis of the cells with 20 pg/ml digitonin.

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kinase C activation has been reported to modulate the subEffects of Phorbol Myristate Acetate on ATP-triggered Sig- sequent metabolism of the inositol polyphosphate products naling Events in Undifferentiated HL.60 Cells-Fig. 1 shows (38, 40) as well as the phospholipase C-catalyzed breakdown the Ca2+-transients elicited by extracellular ATP in control of the polyphosphoinositide,substrates. Fig. 2B illustrates the ) ATPcells and cells pretreated with 100 nM phorbol myristate dose-response relationship (EC50 = 1p ~characterizing acetate (PMA) for 2 min prior to ATP addition. Under these induced InsPs accumulation in control HL60 cells. In cells conditions, PMA did not affect basal cytosolic [Ca"]. Con- pretreated for 3 min with 100 nM PMA, this dose-response versely, the ability of ATP, at concentrations below the nor- relationship was described by a modest shift in ECso to 3 p~ mal EC60, to trigger changes in cytosolic [Ca"] was completely and a 40% decrease in the maximal extent of accumulation. inhibited in these cells (Fig. 2 A ) . With the addition of ATP Thus, both the potency and efficacy of ATP as an activator in the 1-10 p~ range, the PMA-treated cells produced Ca2+ of InsP, accumulation were significantly decreased. These transients, but these were attenuated (relative to those ob- changes in ATP-induced InsP3 production can at least parserved in control cells) with regard to both magnitude and tially explain the 50-fold decrease in potency characterizing duration (Fig. 1). Half-maximal inhibition (IC,) was pro- ATP-triggered Ca2+mobilization (Fig. 2 A ) . The PMA-treated duced by approximately 1 nM PMA. Inhibition of ATP- cells were also characterized by a significant reduction in induced Ca2+mobilization by maximally effective concentra- InsPp accumulation; this latter parameterprobably reflects a tions of PMA (>lo nM) was produced very rapidly, with a tlB combination of both direct PLC-catalyzed PIP breakdown < 10 s. These effects of PMA were mimicked by 4-/3-phorbol and the catabolism of proximally produced InsPB isomers. Not dibutyrate (IC60 = 100 nM), but not by 4-a-phorbol-dideca- surprisingly, there appears to be considerable signal amplifinoate or 4-a-phorbol at concentrations as high as 3 p M (data cation in the ATP-triggered cascade leading from enhanced not shown). Similar inhibitory effects were also observed upon InsPs accumulation to the more distal Ca2+mobilization. In incubation of the HL60 cells with micromolar concentrations control cells, the submaximal %fold increase in InsP3 elicited of two synthetic diacylglycerols, dioctanoylglycerol and by 1 p~ ATP was sufficient to trigger maximal Caz+mobilioleoyl-acetyl-glycerol (data not shown). The dose-response zation. Thus, any pharmacological manipulation must limit relationship (Fig. 2 A ) describing the effects of ATP on cyto- InsP3 accumulation to less than a %fold net increase in order solic [Ca"] in PMA-treated HL60 cells was characterized by to produce an observable attenuation of Ca2' mobilization. Effects of Fluoride and Pertussis Toxin on ATP-triggered a 50-fold increase in E C ~ to O 7.5 p~ (from the control value Signaling Events in Undifferentiated HL60 Cells-Previous O f 150 nM). A rapidly expanding body of observations has implicated studies have demonstrated that phospholipase C activity in protein kinase C in the modulation of several reactions in- intact neutrophils (41, 42) and other cell types (43) can be volved in Ca2+homeostasis (2,3);these include indirect effects activated, in the absence of receptor agonists, by incubation via modification of inositol phosphate metabolism (38) and of the cells in the presence of millimolar concentrations of effects on Ca2+transport/homeostatic mechanismsperse (39). fluoride. These conditions trigger a slow but sustained proThus, at least one effect underlying the ability of PMA to duction of the various inositol phosphates. Sustained accuinhibit ATP-induced Ca2+mobilization might be attenuation mulation of 1,4,5-InsP3 can be accompanied by mobilization of the phospholipase C-catalyzed production of those inositol of Caz+ from intracellular stores; in general, this fluoridephospholipid-derived signaling molecules (particularly, 1,4,5- induced Ca2+mobilization is much more gradual than that IP3) involved in regulation of cellular Ca" fluxes. Protein elicited by receptor agonists. Given the well-characterized RESULTS

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FIG. 1. Inhibition of ATP-induced Ca2+ transients in undifferentiated HL60 cells treated with phorbol ester. Cytosolic [Ca'+]in undifferentiated HL60 cells loaded with fura2 was measured as described under "Materials and Methods." All measurements were made at 37 "C; each transient was recorded using a separate aliquot of 6 X lo6 cells suspended in 1.5 ml of balanced salt solution supplemented with 1 mg/ml bovine serum albumin. Each transient labeled I was recorded from cells treated only with the indicated [ATP]; each transient labeled 2 was recorded from cells which had been pretreated with lo" PMA for 2 min prior to addition of the indicated [ATP]. The transients induced by a given [ATP] in control and PMA-treated cells are graphically superimposed for comparison. Thesetransients, recorded from one cell preparation, were qualitatively and quantitatively representative of those recorded using eight other cell preparations.

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FIG. 2. Dose-response relationships characterizing ATP-induced changes in cytosolic [Ca2+]and inositol polyphosphate accumulation in control and phorbol ester-treated HL60 cells (undifferentiated). A, ATP-induced Ca2+ transients of the type illustrated in Fig.1were recorded fromundifferentiated HL60 cells pretreated in the absence (0)or presence of 100 nM PMA (H)for 2 min prior to addition of the indicated concentrations of ATP. Peak changes cytosolic in [Ca"] were calculated and plotted uersus [ATP]. Each point represents the mean f standard error of data obtained from four separate experiments.B and C , [3H]inositol-labeled preparationsof undifferentiated HL60 cells were incubated at 37 "C in 0.5-ml aliquots containing 1-2 X 10' cells. Individual aliquots were then treated with the indicated [ATP] for 15 s prior to the quenching with perchloric acid. Radioactivity associated with the InsP3 (ZP3,panel B ) and InsPz (ZPZ,panel C ) fractions was measured as described under "Materials and Methods." Data shows the ATP-induced effects in control cells (0)and in cells pretreated with 100nM PMA (m) for 3 min prior to ATP addition. Eachdata point represents the mean f standard error of four determinations using two separate preparationsof labeled cells. FIG. 3. Effects of fluoride on cytosolic [Cas+]- and ATP-induced changes in cytosolic [Ca2+]in undifferentiated HL60 cells. Cytosolic [Ca"'] was measured in undifferentiated HL60 cells loaded with fura2. Each trace wasrecordedfrom a separatealiquot of 6 X lo5 cells suspended in 1.5 ml of basal salt solution. After a 5-minpreincubation at 37 "C, the cellswere treated with 12.5 mM NaC1, 5 p M A1C13 (trace I ) , 11 mM NaF, 5 p~ AlC13 (trace Z ) , or12.5 mM NaF, 5 p M AlC13 (trace 3 ) . After 67 min, each cell preparation was then pulsed with 300 nM ATP.These data were recorded from a single preparation of cellsbutarerepresentative of that obtained in two additional experiments.

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ability of fluoraluminates to interact with regulatory GTP- intracellular Ca2+. These fluoride-inducedeffects onCa2+ bindingproteins involved inadenylate cyclase regulation, mobilizationsuggest that undifferentiated HL60 express a such results have been interpreted as indirect evidence for a GTP-binding protein capable of activating phospholipase C; role of GTP-binding proteins in the activationof phospholi- such a GTP-binding protein might mediate the activationof pase C. Addition of 12.5 mM fluoride produced, after a 1-min phospholipase C by extracellular ATP.To further investigate lag phase, a sustained 2-fold increase in cytosolic [Ca"] (Fig. this possibility, the effects of extracellularATPonboth 3). Furthermore, subsequent addition of 300 nM ATP triginositol phospholipid breakdown and Ca2+mobilization were geredonly a very attenuatedCa2+transient.Incontrast, characterized in undifferentiated HL60 treated with either addition of an equimolar concentration of NaCl neither ele- pertussis toxin ora combination of pertussis toxin and PMA. vated cytosolic [Ca'+] nor attenuated the Ca2+ transient trig-The cells were incubated with pertussis toxin under conditions gered by the extracellular ATP. Treatment of the cells with (100ng/mlfor 3 h) which havebeenshown to produce lower concentrations (6-11 mM) of NaF did not, itself, in alter complete uncoupling of Net-Leu-Phe receptors from phoscytosolic [Ca"] butdidsubstantiallyattenuatetheATPpholipase C in differentiated HL60cells (19-21). Control cells induced Ca2+mobilization. Thus, both fluoride and extracel- or toxin-treated cells were then incubated with PMA under lular ATP appear to trigger mobilization of a common pool of conditions previously shown (Fig. 2) to substantially attenu-

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ate theaction of extracellular ATP. Fig. 4 illustratesthe Ca2+ transients triggered by extracellular ATP at concentrations , which are half-maximally (100 nM),maximally (1 p ~ ) or supramaximally (10 PM) effective. With 100 nM ATP as a stimulus, only a very attenuated ca2+transient was observed in the pertussis toxin-treated cells; no Ca2+transients were observed when control or toxin-treated cells were preincubated with PMA. With 1 p~ ATP as a stimulus, a 50% reduction in the peak change in cytosolic [Ca"] was noted in the toxin-treated cells. Preincubation of these latter cells with PMA completely inhibited ATP-induced effects. As previously demonstrated (Figs. 1 and 2), the Caz+ mobilization triggered by 1 PM ATP in control cells was substantially, but incompletely, inhibited by exposure to PMA. Finally, with a supramaximal concentration of ATP (10 p M ) as a stimulus, significant, but still submaximal (70% of control), Ca2+transients were observed in the cells treated with either pertussis toxin or PMA. Conversely, only an extremely attenuated Ca2+ transient was triggered in cells treated with both the toxin and thephorbol ester. Treatment of HL60 cells with pertussis toxin also produced significant inhibition of the inositol phosphate accumulation triggered by extracellular ATP. Time course studies(not shown) revealed that the toxin-treated cells were characterized by reductions in both the rate and magnitude of InsP3, InsPp, and inositol monophosphate accumulation elicited by supramaximally effective concentrations (>lo p M ) of ATP. Fig. 5 details the dose-response relationships characterizing the effects of ATP on Ca2+mobilization (Fig. 5A) and the rapid phase of InsP3 (Fig. 5 B ) and InsP2 (Fig. 5 C ) accumulation in these pertussis toxin-treated cells, as well as in cells treated with both pertussis tocin and PMA. ATP-induced Ca2+mobilization in toxin-treated cells was primarily altered with respect to potency as the ECb0 was shifted by one log unit to 1.5 PM. There was also a small decrease in efficacy since maximally active concentrations of ATP (>lo p M ) produced peak changes in [Ca"] which were 20-25% lower than those elicited in control cells. In contrast, the potency (Fig. 5 B ) characterizing ATP-induced InsP3 accumulation was unaffected (EC5o = 1 p~ for both control and toxintreated cells). There was,however, a 60% decrease in the amount of InsP3 accumulated in response to maximally activating concentrations of ATP. In addition, ATP-induced InsP2 accumulation (Fig. 5C) in toxin-treated cells was reduced by >80% even at the highest (300 & concentrations of ATP tested. Treatment of the cells with both pertussis toxin and PMA PTx

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produced inhibitory effects on ATP-induced Ca2+ mobilization, InsP3accumulation, and InsP2accumulation which were much more pronounced than those produced by either agent alone (Figs. 2 and 5). In these cells, the effects of maximally activating concentrations of ATP (up to 300 p ~ on) Ca2+ mobilization, InsP3 accumulation, and InsPp accumulation were inhibited by 85,80, and >go%, respectively. EC50 values could not be reliably estimated from the dose-response curves describing Ca2+mobilization and IP2accumulation; the ECso value characterizing ATP-induced InsP3 accumulation (10 PM) was right-shifted one log unit from that measured in both control and toxin (only)-treated cells. Thus, these experiments strongly suggest that a pertussis toxin-sensitive protein plays a role in mediating the transmembrane signaling actions of extracellular ATP in undifferentiated HL60 cells. However, the toxin appears to produce only a partialinhibition of the ATP-triggered actions. Several possible explanations for this phenomenon were tested in the studies described below. Comparison of the Inhibitory Effects of Pertussis Toxin on fMet-Leu-Phe- and ATP-induced Signaling Events in Differentiated HL.60 Cell.-The most obvious explanation for the observed partial inhibition is that theexperimental conditions routinely employed for treatment of the cells with the toxin (100ng/ml for 3 h) resulted in incomplete ADP-ribosylation of the relevant GTP-binding protein. However, treatment of the cells (under tissue culture conditions) with up to 500 ng/ ml pertussis toxin for 24 h produced no additional inhibitory effect on ATP-mediated Ca2+mobilization or InsPs accumulation (data not shown). While ADP-ribosylation of HL60 membrane proteins was not directly measured in this study, previous investigators have used similar incubation conditions to produce: 1) complete inhibition of Met-Leu-Phe-induced signaling events in both differentiated HL60 cells and in freshly isolated human neutrophils (19-23), and 2) complete inhibition of pertussis toxin-catalyzed [32P]NADincorporation into membranes subsequently isolated from neutrophils or HL60 cells (30). To ascertain these results, the effects of pertussis toxin on ATP- and Met-Leu-Phe-triggered signaling events were directly compared in the several preparations of differentiated HL60 cells. Ca2+transients were monitored in control cells, cells treated with 100 nM PMA for 3 min, cells treated with 100 ng/ml pertussis for 3 h, and cells treated with both toxin and phorbol ester; each group of cells was then stimulated with various concentrations of ATP or Met-Leu-Phe. The dose-response relationships describing these agonist-induced changes in cy-

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FIG. 4. ATP-induced CaZ+transients in undifferentiated HL60 cells treated with pertussis toxin. Parallel samples from a single culture of undifferentiated HL60 cells were preincubated for 3 h in the absence or presence of 100 ng/ml pertussis toxin (PTr). Each cell sample was then identically processed in parallel for measurement of cytosolic [Ca2+].ATP-induced Ca2+transients in the control and toxin-treated cells were recorded after no additional treatment ( I ) orafter an additional 2-min exposure to 100 nM PMA. These data are representative of four separate experiments.

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FIG. 5. Dose-response relationships characterizing ATP-induced changes in cytosolic [Ca"] and inositol polyphosphate accumulation in control, pertussis toxin-treated, and pertussis toxin plus phorbol ester-treated HL60 cells (undifferentiated).A, ATP-induced Ca2+transients of the type illustrated in Fig. 5 were recorded from control undifferentiated HL60 cells (O), cells treated 3 h with 100 ng/ml pertussis toxin (A),or toxin-treated cells additionally exposed to 100 nM PMA (W) for 2 min prior t o addition of the indicated concentrationsof ATP. Peak changes in cytosolic [Ca"] were calculated and plotted uersw [ATP].Each point represents the mean & standard error of data obtained from four separate experiments. B and C, parallel samples of [3H]inositol-labeledpreparations of undifferentiated HL60 cellswere incubated in the absence or presence of 100 ng/ml pertussis toxin for 3 h.Each fraction was then washed, distributed in0.5-ml aliquots containing 1-2 X lo6 cells, and incubated at 37 "C. Individual aliquotswere then treated with the indicated [ATP] for 15 s prior to the quenching with perchloric acid. Radioactivity associated with the InsP3 ( P 3 , panel B ) and InsPz (ZPz,panel C) fractions was measured as described under "Materials and Methods." Data shows the ATPinduced effects in control cells (O),pertussis toxin-treated cells (A),and in pertussis toxin (PTx)-treated cells additionally pretreated with 100 nM PMA (W) for 3 min prior to ATP addition. Each data point represents the mean & standard error of data derived from three separate experiments. 2400

FIG. 6. Dose-response relationshipscomparingtheinhibitoryeffects of pertussis toxin treatmentor phorbolestertreatment on ATPand Ca2+ transients in differentiated HL60 cells. Peakchangesincytosolic [Ca"] were calculated from the tram sients. The dose-responserelationships characterizing the response to ATPor Met-Leu-Phe (FMLP) are shown for untreated cells (O), pertussis toxin (PTx)-treated cells (U), PMA-treated cells (O), and cells treated with both pertussis toxin and PMA (0).These data are representative of that obtained in three separate experiments.

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response relationship was characterized by less than a single tosolic [Ca"] are detailed in Fig. 6. As in undifferentiated HL60 cells, treatment of the differentiated cells with either log unit shift inEC50(100 nM uersw the 20 nM control value). pertussis toxin alone or PMA alone significantly inhibited theConversely, Ca2+mobilization in response to supramaximal Ca2+mobilization triggered by submaximal amounts of ATP; concentrations (up to 3 p M ) of Met-Leu-Phe was inhibited in both cases, the inhibition could be overcome upon addition by >95% in the cells treated only with pertussis toxin; no of higher concentrations of ATP (ECSo= 0.1, 0.8, and 8 p~ response could be elicited in cells treated with both the toxin forcontrol,toxin-treated,andPMA-treated cells,respecand the phorbol ester. tively). Once again, treatment with both pertussis toxin and Treatment of the differentiated HL60 cells with pertussis PMA resulted in >85% inhibition of the Ca2+ mobilization toxin also resulted in complete blockade of Met-Leu-Pheelicited by supramaximal concentration of ATP (100 p ~ ) A . inducedInsPaandInsPzaccumulation while producing a different patternof inhibition characterized the effects of the substantial (>70%), but still incomplete, inhibition of the toxin andPMA on Met-Leu-Phe-induced change cytosolic in InsPs/InsPz accumulation triggered by maximally activating [Ca"]. In the PMA-treated cells, the Wet-Leu-Phe doseconcentrations of ATP. In controlcells, a similar efficacy (5-

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Regulation of Inositol Phospholipid Breakdown in HL60 Cells

FIG. 7. Dose-response relationships comparing ATP- and m e t Leu-Phe-induced changes in inositol polyphosphate accumulation in control and pertussis toxin-treated HL60 cells (differentiated). HL60 cells were simultaneously labeledwith [3H]inositol and differentiated by exposure to 0.5 mM dibutyryl cyclic AMP for 60 h. Duplicate portions of these cells were then incubated for an additional 3 h in the absence or presence of 100 ng/ ml pertussis toxin. Individual aliquots (0.5 ml containing lo6 cells) were then treated with the indicated concentrations of ATP (panelA ) or met-Leu-Phe for 15 s prior to quenching with perchloricacid.Radioactivity associated with InsP3 (ZP3, closed symbols) and InsPl (IPz, open symbols) was determined. Data points (average k range of duplicate determinations) represent accumulation measured in control cells (0,0 ) and pertussis toxin-treated cells (m, 0). Similar data were obtained in two additional experiments.

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6-fold increase in 15 s) characterized InsP3 accumulation in response to either ATP or Met-Leu-Phe (data not shown). , not met-Leu-Phe (up to 3 Conversely, ATP (>lo p ~ ) but p ~ ) could , still elicit a %fold increase in InsPa levels in the toxin-treated cells (Fig. 7). Thus, aswas observed in undifferentiated HL60 cells, pertussis toxin treatment produced only a partial inhibition of ATP-mediated signaling events in the differentiated cells. This did not appear to be due to inadequate ADP-ribosylation of target proteins by the toxin since Met-Leu-Phe-triggered signaling was virtually abolished. Moreover, the inhibition of both ATP- and met-Leu-Pheinduced InsP3 accumulation by the toxin was produced over the same range of toxin concentrations (Fig. 8). The response of the cells to either agonist was half-maximally inhibited after a 3-h exposure to 5 ng/ml of the toxin; maximal inhibition was observed in cells treated with >10 ng/ml of the toxin. Significantly, incubation with higher concentrations of the toxin did not eliminate the residual 2-fold increase in InsP3 accumulation elicited by extracellular ATP (Fig. 8). ATP-induced Signaling Events in HL60 Cells Incubated in the Absence of Extracellular Calcium-It was important to rule out a possible effect of ATP-induced Ca2+influx on the pertussis toxin-insensitive activation of inositol phospholipid hydrolysis. This possibility was tested by measuring inositol phosphate accumulation in pertussis toxin-treated HL60 cells incubated in medium containing 4 0 0 nM [Ca"]. Significantly, neither the efficacy (maximal 4.5-fold increase) nor ) the initial rates of the potency (E& = 1p ~ characterizing ATP-stimulated InsPBandInsP2 accumulation (datanot shown) were affected by removal of extracellular calcium. Pertussis toxin treatment had no effect on the potency characterizing the effects of ATP on InsPs accumulation but did produce a 45% decrease in efficacy. Finally, pertussis toxin treatment produced a 75% decrease in the maximum rate of InsP2accumulation. Thus, neitherthe effects of ATP on Ca2+ mobilization and inositol phosphate accumulation nor the partial inhibitory effects of pertussis toxin on these latter parameters was substantially altered in the absence of Ca" influx across the plasma membrane. ATP-induced Ca2+Mobilization in HL60 Cells Pretreated

with Cholera Toxin-Verghese et al. (30) have demonstrated that cholera toxin, as well as pertussis toxin, can catalyze ribosylation of the 40-kDa a-subunit of a GHL/Gc-typeprotein in plasma membranes derived from human neutrophils, undifferentiated HL60 cells, or U937 cells. However,this cholera toxin-catalyzed ribosylation is significantly inhibited by the micromolar levels of guanine nucleotides which are present in thecytosol of intact cells. Thus, while cholera toxin treatment of intact neutrophils produced only partial ribosylation of the 40-kDa subunit, this was sufficient to significantly reduce the interaction of chemotactic peptides with high affinity receptor-binding sites (30). It was of interest, therefore, to determine whether the Ca2+-mobilizingeffects of extracellular ATP were modified in HL60 cells treated with cholera toxin alone or in combination with pertussis toxin. Parallel culturesof undifferentiated HL60 cells were cultured for 3 h in the presence of cholera toxin alone (300 ng/ml), pertussis toxin alone (100 ng/ml), or bothtoxins. The effects of ATP on Ca2+mobilization were then tested in the treated cells and on control sample of untreated cells. The doseresponse curve obtained using the cholera toxin-treated cells was identical to that of the control cells (data not shown). Likewise, the cells cotreated with both toxins exhibited identical dose-response curves, i.e. both cell populations showed the one log unit increase in ECm characteristic of cells treated with pertussis toxin alone (Fig. 5A). DISCUSSION

The results of this study indicate that the HL60 human promyelocytic leukemia cell line is a useful model system for investigation of the molecular mechanisms which mediate activation of the inositol phospholipid signaling system by P2-purinergic receptors for ATP. The mechanism whereby these receptors are coupled to the phospholipase C effector system exhibits several, but not all, of the features which characterize the coupling of chemotactic peptide receptors to the same effector system. Effects of Protein Kinase C Activation-Activation of protein kinase C in intact cells by tumor-promoting phorbol esters and exogenous diacylglycerol analogs has been shown

Regulation of Inositol Phospholipid Breakdownin HL60 Cells relationshipcharacterizingtheinhibitory action of pertussis toxin on ATPand met-Leu-Phe-induced inositol polyphosphate accumulation in differentiated HL60 cells. HL60 cells were differentiated and [3H]inositol-labeled as described under “Materials and Methods.” Six parallel aliquots of the cell preparation were incubated withthe indicated concentrations of pertussis

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toxin for 3 h subdivided at 37 “C. Each then further intoaliquot 0.5-ml was aliquots containing lo6 cells.Each of these was then treated with either 30 p M ATP 0” (O),3 p~ met-Leu-Phe (W), or no agonist (A)for 15 s prior to perchloric acid quenching; radioactivity associated with the InsP3 UP3,left panel) and InsP2( ~ P z , right panel) fractions was determined. Data points represent the mean f standard error of triplicate determinations from a single experiment.

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t o induce partial or complete inhibition of signaling events pool of intracellular Ca2+which is mobilized by the Pz-puritriggered by the majority of Ca2+-mobilizing agonists tested nergic receptors in undifferentiated HL60 cells (Fig. 3) prothus far(reviewed in Refs. 2 and 3). As previously noted, this vides indirect evidence that Ca2+ mobilization in response to occupation of these receptors is mediated by GTP-binding includes the Ca2+ mobilization or InsP3 accumulation trigregulatory proteins. Additional studies with both hepatocytes gered byextracellular ATP in rat hepatocytes (9) and Ehrlich tumor cells (10). Ina similar manner, the abilityof extracel- (43) and human neutrophils (42) have demonstrated that this lular ATP to activate Ca2+ mobilization (Figs. 1 and 2 A ) and Ca2+ mobilization can be correlated with enhanced accumuthe accumulation of InsP3 (Fig. 2B) and InsPz (Fig. 2 C ) in lation of Inspa. The abilityof F- to initiate phosphoinositide HL60 cells was significantly,but not totally, inhibited by brief breakdown in both rat hepatocytes and neutrophils issignifexposure to PMA. That these effects of PMA were due to icant since the G-protein responsibleforphospholipaseC protein kinase C activation was supported by the nanomolar activation is believed to be pertussis toxin insensitive in the ICw characterizing the PMA action, the rapid onsetof inhi- hepatocytes (43, 44), butpertussistoxinsensitiveinthe neutrophils (22, 23). Thus, F- should activate thephospholibition,andtheability of othertumor-promotingphorbol estersand exogenous diacylglycerol analogs t o mimic the pase in undifferentiated HL60 cells regardless of which GPMA-induced inhibition. Theseeffects were also observed in protein subtypes may be expressed. Both undifferentiated (27, 29, 30) and differentiated (28, HL60 cells differentiated along the neutrophil pathway(Fig. 6 A ) .Significantly, allof these characteristicsof protein kinase 29) HL60 cellshave been shown to express the pertussis C-induced inhibition of ATP-triggered signaling in the HL60 toxin-sensitive G-proteinbelieved to couple chemotactic pepcell line are very similar to those reported by Kikuchi et al. tide receptors to the PI-PLC effector system in phagocytic leucocytes. Since undifferentiated HL60 do not express sig(15) in a study of chemotactic peptide-induced signaling in nificant numbers of chemotactic peptidereceptors, studies of differentiatedHL60 cells. Theseworkersalsonotedthat phorbol ester treatment produced a significant, but incom- the role of this G-protein in receptor-phospholipase C couplete, inhibition of Met-Leu-Phe-induced InsP3 accumulapling have been limited to HL60 cells induced to differentiate tion. Significantly, the major effect was a 50% decrease in the by either dimethyl sulfoxide or dibutyryl cyclic AMP (16, 19maximal rate of InsP3 accumulation.A similar partial inhib- 21). However, our finding that undifferentiated HL60 cells itory actionof Met-Leu-Phe-induced InsPs accumulation hasalso express P2-purinergic receptors which are functionally been measuredinhumanneutrophils(17).Wehave also coupled to phospholipase C has permitted a functional evalobserved that exposure of differentiated HL60 cells to PMA uation of this pertussis toxin-sensitive G-protein in the nonsignificantlyreduces the potency, but not the efficacy, of induced cells. The most interesting feature of these experiMet-Leu-Phe in triggering Ca2+mobilization (Fig. 6B). Re- ments was that pertussis toxin treatment produced only a cent studies by Kikuchi et al. (16) on differentiated HL60 partial inhibitionof the inositolphospholipid signaling events cells and by Smith et al. (17) on human neutrophils have triggered by occupation of the ATP receptors. Using InsP3 indicated that the inhibitory effects of protein kinase C on accumulation a t 15 s as the most proximalindex of receptorthe coupling of chemotactic peptide receptors to the phospho- phospholipase C coupling, we compared the dose-response lipaseCeffector system may be primarilymediated by a relationships in control and toxin-treated cells (Fig. 5B). In kinase-induced modification of the G-protein/phospholipase both cell populations, ATP had the same potency (E& = 1C interaction rather the receptorjG-protein interaction. It is 2 p M ) but its efficacy was reduced by 60%. Thus, even at very highly likely that this mechanismis at least partially respon- high receptor occupancy there was significant inhibition of sible for the inhibition of ATP-induced activation of InsPs ATP-induced polyphosphoinositide hydrolysis. It should be accumulation in HL60cells. noted that the radioactivity contained in our InsP3 fractions Effects of Pertussis Toxin and the Role of GTP-binding measured at 15s undoubtedly represents countsderived from Proteins-The ability of fluoroaluminates to modulate the a mixture of InsP3 andinositol tetrakisphosphate isomers as function of GTP-binding proteins has been extensively doc- well as acid-hydrolyzed forms of cyclic InsP3 which may also umented (4).The ability of NaF toinduce release of the same be formed (45-47). Thus, the actual percentages of 1,4,5-InsP3

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Regulation of Inositol Phospholipid Breakdown in HUO Cells

may be somewhat different in the ‘‘InsPC fractions derived from control and pertussis toxin-treated cells. The inhibitory effects of pertussis toxinwere also apparent inmeasurements of the ATP-induced Ca2+mobilization (Figs. 4 and5A). Here the net effect was a one log unit increase in ECSofrom the control vaolue of 150 nM ATP. These results indicate that, with submaximally activatingconcentrations of ATP, the toxin-induced attenuation of 1,4,5-InsP3 accumulation was sufficient to reduce mobilization of the 1,4,5-InsPs-releasable Ca2+stores. However, the residual, toxin-resistant 1,4,5-InsP3 accumulated in response to higher doses of ATP was sufficient to induce near maximal Ca2+mobilization. The results of several experiments indicated that thepartial suppression of ATP-induced signaling in either undifferentiated or differentiated HL60 cells was not due to incomplete ribosylation of pertussis toxin substrates. In particular, the responses of differentiated cells to chemotactic peptide stimulation were used to assess the effectiveness of the toxin treatment regimen in producing complete functional modification of the relevant G-protein. This functional modification appeared to be complete since Met-Leu-Phe-induced Ca2+ mobilization (Fig. 6B) and InsP3 accumulation (Figs. 7 and 8) was completely abolished. In these differentiated HL60 cells, the dose-response relationships showed that both ATPand Met-Leu-Phe-induced InsP3/InsPz accumulation were equally sensitive to pertussis toxin treatment (Fig. 8). While the toxin was equipotent in inhibiting the actions of both receptors, its 100% efficacy in inhibiting the Met-Leu-Phe effects contrasted with its 70% efficacy in attenuating the ATP-induced signaling events. Several possible mechanisms may underlie the residual ATP-induced activation of inositol phospholipid breakdown in pertussis toxin-treated HL60 cells. The first possibility is based on the assumption that HL60 cells express only one Gprotein which is coupled to the PI-PLC. That is, both chemotactic peptide receptors and the Pz-purinergic receptors would induce activation of PI-PLC only via interaction with the pertussis toxin-sensitive GHL/GCprotein (27, 28). In this case, however, ribosylation of the 40-kDa a-subunit would be insufficient to completely block coupling between P2-purinergic receptors and PI-PLC. Thus, it may be possible that while ribosylation results in de facto prevention of the interaction of GHL/Gc with chemotactic peptide receptors, it may induce only a destabilization of the interaction with PZpurinergic receptors. A second possible mechanism is based on the assumption that HL60 cells can express two distinct G-proteinswhich are functionally coupled to the PI-PLC but that only one, i.e. GHL/Gc, is a pertussis toxin substrate. Given this model, the existing data would suggest that the chemotactic peptide receptor can only interact with GHL/Gc. Conversely, the Pppurinergic receptors can interact with both GHL/GCand the other pertussis toxin-insensitive G-protein. Results from a number of studies provide at least circumstantial support for this possibility. In hepatocytes, Pz-purinergic receptors have been shown to activate inositol phospholipid breakdown via a pertussis toxin-insensitive mechanism (11, 12). We have also demonstrated that ATP activates Ca2+mobilization and InsP3 accmumulation with equal potency and efficacy in both control and pertussis toxin-treated DDT1 smooth muscle cells.3Thus, thereis ample evidence that P2-purinergic receptors similar (based on nucleotide specificity) to those expressed in HL60 cells can activate phospholipase C via pertussis toxin-insensitive mechanisms. The question remains whether HL60 cells do indeed express pertussis toxin-insenG. R. Dubyak, unpublished observations.

sitive G-proteins which are coupled to phospholipase C. Uhing et al. (27) have recently isolated a number of GTP-binding proteins from undifferentiated HL60 cells. While these include G, and two pertussis toxin substrates, therewas a major GTPyS-binding protein with a molecular mass of about 23 kDa. Finally, the possibility should be considered that the pertussis toxin-insensitive mobilization of intracellular Ca2+ stores by ATP may be mediated by some mechanism alternative to the InsP3-mediated release process. The ability of GTP perse to induce Ca2+release from subcellular stores has been well documented (49), although the physiological significance of this action in intact cells remains unknown. It is also interesting to note that Ca2+transients can still be elicited by leukotriene B4 and platelet-activating factor in pertussis toxin-treatedhuman neutrophils under conditions where Met-Leu-Phe-inducedtransientsare completely blocked (23). Effects of Combined Pertussis Toxin Treatment and Protein Kinase C Activation-Given the substantial, but partial, inhibition of ATP-induced signaling eventsin HL60 cells treated with either pertussis toxin or activators of protein kinase C, it was of interest to characterize the signaling actions of extracellular ATP in cells pretreated with both inhibitory agents. In PMA-treated cells (Fig. ZB),the maximal InsP3 accumulation was 60% of the control value while in pertussis toxin-treated cells (Fig. 5B) the same parameter was 40%of control; in cells treated with both agents(Fig. 5B) maximal InsPs accumulation was about 20-25%of control. The lack of an additive effect strongly suggests that thetwo inhibitory conditions are not affecting separate, but parallel, reactions in the ATP-mediated signaling cascades. The results can be accommodated within either the “single” or “multiple” G-protein models described above if one assumes that the effects of protein kinase C are: 1) mediated at thelevel of Gprotein-phospholipase C coupling, and 2) the kinase can affect the activation of the phospholipase C by eitherpertussis toxin-sensitive or insensitive GTP-binding proteins. In contrast to these actions on InsP3 accumulation, the combined effect of treatment with pertussis toxin and protein kinase C activators was synergistic when Ca2+mobilization was the measured parameter. In either undifferentiated (Figs. 4 and 5A) or differentiated (Fig. 6) HL60 cells treated with both agents, ATP-induced Ca2+mobilization was drastically inhibited even at the highest agonist concentrations employed. The ability of protein kinase C to almost abolish Ca2+ mobilization in thepertussis toxin-treatedcells while exerting only an additional 40% inhibition of nominal InsP3 accumulation (as compared to cells treated with toxin alone) underscores the probable action of protein kinase C in modulating several reactions in the transduction pathway from the Pzpurinergic receptors to Caz+mobilization. This may include activation of Ca’+-homeostatic mechanisms such asthe plasma membrane CaZ+-ATPasepump (49) or activation of 1,4,5-InsP3 metabolism (38). In summary, we have presented evidence indicating that Ps-purinergic receptors may activate the polyphosphoinositide-phospholipase C in HL60 cells via the mediation of a pertussis toxin-sensitive GTP-bindingprotein, which also mediates the actions of chemotactic peptide receptors in these and other phagocytic white blood cells. However, our data also suggest that these same receptors can be coupled to the phospholipase via an additional pertussis toxin-insensitive mechanism. Thislatter finding raises the possibility that undifferentiated HL60 cells express two distinct GTP-binding proteins coupled to phospholipase C; one of these is very

Regulation of Inositol Phosphollipid Breakdown inHL60 Cells likely to be the GHL/GCprotein recently isolated from this cell line. Significantly, the data of Oinuma et al. (28) and Falloon et al. (29) indicate that expression of the 40-kDa asubunit/toxinsubstrate increases upon differentiation of HL60 cells along the granulocyte pathway. It would be interesting to determine whether expression of the putative, pertussis toxin-insensitive G-protein decreases with differentiation of these and other myelo-monocytic progenitor cells. Such studies should be facilitated by the fact that the Pzpurinergic receptors are expressed in all human myelopoietic cells from the late myeloblast stage through the terminally differentiated stages represented by circulating neutrophils and monocytes. REFERENCES Gordon, J . L. (1986) Biochem. J. 233, 309-319 Abdel-Latif, A.A. (1986) Pharmacol. Rev. 3 8 , 228-272 Berridge, M. J. (1987) Annu. Rev. Biochem. 5 6 , 159-193 Gilman, A. G. (1987) Annu. Reu. Biochem. 56,615-650 Cockcroft, S. (1987) Trends Biochem. Sci. 1 2 , 75-79 Lad, P. M., Olson, C.V., Grewal, I. S., and Scott, S. J . (1985) Proc. Natl. Acad. Sci. U. S. A. 82,8643-8647 7. Molski, T. F. P., Naccache, P. H., Marsh, M. L., Kermode, J., Becker, E. L., and Sha’afi, R. I. (1984) Biochem. Biophys. Res. Commun. 124,644-650 8. Nakamura, T., and Ui, M. (1985) J. Bioi. Chem. 260,3584-3593 9. Charest, R., Blackmore, P. F., and Exton, J. H. (1985) J. Biol. Chem. 260,15789-15794 10. Weiner, E., Dubyak, G., and Scarpa, A. (1986) J. Biol. Chem. 261,4529-4534 11. Irving, H. R., and Exton, J. H. (1987) J. Biol. Chem. 2 6 2 , 34403443 12. Okajima, F., Tokumitsu, Y., Kondo, Y., and Ui, M. (1987) J. Biol. Chem. 262, 13483-13490 13. Collins, S. J., Gallo, R. C., and Gallagher, R. E. (1977) Nature 270,347-349 14. Collins, S. J. (1987) Blood 70, 1233-1244 15. Kikuchi, A., Kozawa, O., Hamamori, Y., Kaibuchi, K., and Takai, Y. (1986) Cancer Res. 4 6 , 3401-3406 16. Kikuchi, A., Ikeda, K., Kozawa, O., and Takai, Y. (1987) J. Biol. Chem. 262,6766-6770 17. Smith, C.D., Uhing, R. J., and Snyderman, R. (1987) J. Bioi. Chem. 262,6121-6127 18. Naccache, P. H., Molski, T. F. P., Borgeat, P., White, J. R., and Sha’afi, R. I. (1985) J. Biol. Chem. 260, 2125-2131 19. Brandt, S. J., Dougherty, R. W., Lapetina, E. G., and Niedel, J. E. (1985) Proc. Natl. Acad. Sci. U. S. A . 82,3277-3280 20. Anthes, J. C., Billah, M. M., Cali, A., Egan, R. W., and Siegel, M. I. (1987) Biochem. Biophys. Res. Commun. 145,825-833 21. Kikuchi, A,, Kozawa, O., Kaibuchi, K., Katada, T., Ui, M., and Takai, Y. (1986) J. Biol. Chem. 2 6 1 , 11558-11562 1. 2. 3. 4. 5. 6.

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