THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1992 by The American Society for Biochemistry and Molecular Biologv, Inc.

Vol. 267, No. 10, Issue of April 5 , pp. 660-6610, 1992 Printed in U.S.A.

Dual Regulation of Arachidonic Acid Release by P2u Purinergic Receptors in Dibutyryl Cyclic AMP-differentiated HL60 Cells*

(Received for publication, March 15, 1991, and in revised form, September 4, 1991)

Mingzhao Xing$, Frank Thevenodi, and Rafael Matterall From the Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neu- trophil-like HL60 cells: stimulation in the micromolar range (ECao = 3.2 f 0.9 PM) and inhibition at higher concentrations (ECeo = 90 f 11 PM). ATP also inhibited UTP- and platelet activating factor-stimulated AA re- lease. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca”, cell permeabilization, or changes in the rise of [Ca2+Ii induced by agonist. The inhibition was rapid, being detected within 6-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desen- sitized by pretreatment of the cells with 2 mM ATP, but not 20 p~ ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on genera- tion of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with PI purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5’-(BYy-methylene)triphosphate (AMP-PCP), adenyl-S’-yl imidodiphosphate (AMP- P(NH)P), ADP, adenosine 5’-0-(3-thiotriphosphate) (ATPrS), and UTP), only UTP and ATPrS displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EGO = 60-300 pM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of Pz” receptors in HL60 cells.

Extracellular adenosine compounds interact with mem- brane-bound purinergic receptors to influence biological proc- esses such as platelet aggregation, neurotransmission, cardiac function, muscle contraction, and liver glycogen metabolism (for review see Ref. 1). Based on pharmacological criteria, these receptors are classified into PI and Pp types (2). The PI-

* This work was supported in part by Diabetes Association of Greater Cleveland Grant 326-89, American Heart Association (North-

BRSG S07RR-05410-28, Ohio Board of Regents Grant RIFOBR, east Ohio Affiliate) Grant 4719, National Institutes of Health Grant

and CRC/American Cancer Society Grant IRG-186 (to R. M.). 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 solely to indicate this fact.

$ This work was performed in partial fulfillment of the research requirements corresponding to the departmental Ph.D. program.

5 Recipient of a fellowship from the Max-Kade Foundation. 7 To whom correspondence should be addressed Dept. of Physi-

ology and Biophysics, Case Western Reserve University School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106. Tel.: 216-368- 3180; Fax: 216-368-5586.

type is preferentially occupied by adenine compounds in the following order of potency: adenosine > AMP > ADP > ATP; the reverse applies to the Pz receptors (1). Different subtypes of Pz purinoceptors have been defined, based on relative agonist potency, including Ppx (order of potency: AMP-CPP > AMP-PCP > ATP = 2MeSATP) and Ppy (2MeSATP >>

This classification extends to include two distinct cell-specific subtypes: the PZT and P2z receptors expressed in platelets and mast cells, respectively (1). The nucleotide receptors ex- pressed in some cell types, including HL60 cells and neutro- phils, recognize ATP and UTP with similar affinities (3-9) and cannot be classified within the previously mentioned subtypes. This led to the two recently proposed nomenclatures classifying them as either “Pzu” purinergic receptors (10) or “nucleotide” receptors (ll), the latter reflecting the equipo- tency of a purine and a pyrimidine.’

Recent studies have defined intracellular signaling systems coupled to Pz purinergic receptors. Among them, the regula- tion of PLC’ activity and intracellular Cap+ mobilization are the best characterized, especially in neutrophils and HL60 cells (3-a), turkey erythrocytes (12), and hepatocytes (13,14). Pz purinergic receptors are also coupled to stimulation of AA release in neutrophils and HL60 cells (6), astrocytes (15), and Chinese hamster ovary cells (16). At least in HL60 cells, this process seems to result from the direct interaction between Pp purinergic receptor, G protein, and PLAZ (6). Other effec- tors regulated by Pz purinergic receptors include PLD in hepatocytes (17) and HL60 cells (la), adenylyl cyclase in hepatocytes (14), and guanylyl cyclase in FRTL5 cells (19).

In general, occupancy of Pz purinergic receptors increases the production of intracellular second messengers. Exceptions to this rule are the inhibition of agonist-stimulatedproduction of CAMP in hepatocytes (14) and cGMP in FRTL-5 thyroid cells (19). We now report that ATP and UTP exert a dual regulation of the release of AA in dibutyryl CAMP-differen- tiated HL60 cells, consistent with their interaction with two different Pzu receptor subtypes.

ATPyS > ATP = ADP > AMP-CPP > AMP-PCP = UTP).

The P2u nomenclature is used in this article. The abbreviations used are: PLC, phospholipase C; PLAz, phos-

pholipase AZ; PLD, phospholipase D; AA, arachidonic acid; IPz, inositol 4,5-bisphosphate; IP,, inositol 1,4,5-trisphosphate; fMLP, N - formyl-methionyl- L-leucyl-L-phenylalanine; HEPES, 4-(2-hydroxy- ethyl)-1-piperazineethanesulfonic acid; EGTA, [ethylene- bis(oxyethylenenitrilo)]tetraacetic acid; IMDM, Iscove’s modified Dulbecco’s medium; HBSS, Hank’s balanced salt solution; BtZcAMP, dibutyryl cyclic adenosine 3’:5’-monophosphate; AMP-P(NH)P, ad- enyl-5”yl imidodiphospate; ATPyS, adenosine 5’-0-(3-thiotriphos- phate); 2MeSATP, 2-methylthioadenosine 5”triphosphate; AMP- CPP, adenosine 5’-(a&methylene)triphosphate; AMP-PCP, adeno- sine 5’-(fi,y-methylene)triphosphate; [Ca2+Ii, cytosolic free Ca2+; XTP, xanthosine 5”triphosphate.

6602

Dual Regulation of AA Release by P2 Purinoceptors 6603

EXPERIMENTAL PROCEDURES

Materials-ATP, ADP, AMP, XTP, BbcAMP, mepacrine, aden- osine deaminase, fMLP, arachidonic acid and the insulin-transferrin- sodium selenite supplement for culture media were obtained from Sigma. UTP, GTP, CTP, TTP, AMP-P(NH)P, AMP-PCP, and ATPrS were purchased from Boehringer Mannheim. ~-rnyo-[2-~H] Inositol was obtained from American Radiolabeled Chemicals, Inc. and [5,6,8,9,11,12,14,15-3H]arachidonic acid from Du Pont-New Eng- land Nuclear. IMDM was from GIBCO. Bovine calf serum was from HyClone Laboratories. Fura-S/AM was obtained from Molecular Probes, Inc. Phospholipid standards were purchased from Avanti Polar Lipids and Serdary.

Culture and Differentiation of HMO Cells-HL6O cells (originally obtained from the American Type Culture Collection and kindly provided by Dr. G. Dubyak, Case Western Reserve School of Medi- cine) were routinely grown in IMDM supplemented with 25 mM HEPES, pH 7.4, 35 mM NaHC03, and 10% calf bovine serum. Cells were cultured under a 92.5:7 5 air:CO, atmosphere, at a cell density of 0.2-1.0 X lo6 cells/ml. Beiore differentiation, cells were diluted to approximately 0.5 X lo6 cells/ml and grown for 12-24 h in serum- free IMDM supplemented with 5 pg/ml insulin, 5 p:/ml transferrin, 5 ng/ml sodium selenite, 100 units/ml penicillin, :nd 100 pg/ml streptomycin (IMDM with defined supplements), until a cell density of 0.8-1 X lo6 cells/ml was reached. Differentiation into a neutrophil- like phenotype was induced at this point by addition of 0.5 mM BtpcAMP and culture for 72 h. Only cells with up to 30 passages were used throughout this study. These culture conditions were considered optimal for differentiation and expression of fMLP-promoted effects.

Release of Arachidonic Acid in HL60 Cells-Approximately 0.5-1 X 10' cells/70 assay tubes were differentiated, as indicated in the previous section. At the end of differentiation, cells were harvested by centrifugation (5 min at 300 X g and room temperature), resus- pended in IMDM with defined supplements (see previous paragraph) a t a concentration of 3.5 X lo6 cells/ml, and labeled at 37 "C for 1.5 h with 1 pCi/ml [3H]arachidonic acid (specific activity = 76 Ci/mmol, 13 nM final concentration). Labeling was very rapid, and steady state was reached after approximately 60 min; under these conditions, more than 70% of the total added precursor is incorporated into the phospholipid cellular fraction. The major labeled species were phos- phatidylcholine, phosphatidylinositol, and phosphatidylethanola- mine. At the end of the isotopic labeling, cells were washed twice with ice-cold HBSS containing 0.8 mM MgC12, the indicated concentration of CaCl,, 0.1% glucose, and 0.3% protease- and fatty acid-free bovine serum albumin (supplemented HBSS). Cells were then resuspended at 6 X lo6 cells/ml in the same solution further supplemented with 0.4 mM unlabeled AA and kept on ice. Assays were started by adding 200 p1 of the packed labeled cell suspension to 200 pl of supplemented HBSS (prewarmed at 37 "C), containing stimuli. Incubations were carried out at 37 "C for 8 min, under constant shaking. Reactions were stopped by 10-fold dilution with ice-cold 50 mM Tris/HCl, pH 7.5, 100 mM KCl, 5 mM EGTA, 5 mM EDTA, followed by centrifu- gation for 20 min at 1400 X g and 4 "C. Two-ml aliquots of the supernatants were added to 10 ml of scintillation fluid and counted. We have validated, by extraction of supernatants with chloro- form:methanol followed by concentration and separation on TLC plates, that the counts released into the supernatant indeed represent ["Hlarachidonic acid. Under these assay conditions, 2 p~ fMLP or 15 p M ATP promoted the release of up to 10% of incorporated isotope after 8-10 min of stimulation.

PLC Assays in HL60 Cells-Undifferentiated HL60 cells (0.8 X IO6 cells/ml), previously grown for 12-24 h in serum- and inositol-free IMDM with defined supplements (see above), were combined with 0.5 mM BtZcAMP and 2 pCi/ml of [3H]inositol (specific activity = 15 Ci/mmol, 133 nM final concentration). Cells were then differentiated and labeled simultaneously by incubation under these conditions for 72 h at 37 "C (3). At the end of this period, cells were washed twice and resuspended at 6 X lo6 cells/ml in supplemented HBSS (see previous paragraph) also containing 10 mM LiCl (to inhibit inositol phosphate and IP, phosphatases). Assays were started by the addition of 100 p1 of the cell suspension to an equal volume of supplemented HBSS prewarmed at 37 "C and containing test substances. Incuba- tions were carried out for 8 min at 37 "C, thereby measuring the maximal accumulation of inositol phosphates promoted by agonists (3). Reactions were stopped by the addition of 1 ml of chloro- form:methanol:HCl (200:1001, by volume) followed by 200 pl of 125 pg/ml phytic acid hydrolyzate. Tubes were vortexed and centrifuged for 10 min at 1400 X g and room temperature. Aliquots (550 p1) of

the aqueous (upper) phases were collected, diluted to 10 ml with H,O and loaded on top of AGl-X8 (chloride form) columns (2-ml bed volume) (20). The loaded columns were first run with 10 ml of 30 mM HCl, to elute myo-inositol and monophosphorylated inositols. [3H] IP, and [3H]IP3 were coeluted with 3 ml of 0.5 M HCl, mixed with 15 ml of scintillation liquid, and counted.

Extraction of Cellular Lipids and Their Separation by TLC-Total cellular lipids were extracted according to Bligh and Dyer (21). Cell suspensions or supernatants were first mixed with chloro- form:methanol:water (1:2:0.8, by volume). After 10 min, additional chloroform and water were added to create a final volume ratio of 1:l:O.g. The lipid phase was collected and dried under vacuum. The total lipid residues were then dissolved in 50 pl of chloro- form:methanol:water (75:25:2, by volume), and 40-pl aliquots were spotted on 20 X 20-cm silica gel plates. Plates were subjected to ascending chromatography using as solvents either chloro- form:methanol:acetic acidwater (75:50:106, by volume), for separa- tion of phospholipids (22), or ethyl acetate:water:isooctane:acetic acid (45:5025:10, by volume), for the separation of free fatty acids (23). Standards were simultaneously run on each plate and visualized using iodine vapor.

Fluorescence Microscopy-Differentiated HL60 cells, prepared as described above, were incubated at 37 "C for 8 min in the presence of 2 p~ propidium iodide (668 Da) either under conditions identical to those used when studying the nucleotide effects on AA release or in the absence of added calcium and magnesium salts and presence of 5 mM each of EGTA and EDTA. Addition of 50 p~ digitonin was used as a positive control for permeabilization. Cells were subsequently examined under a fluorescence microscope.

Measurement of Cytosolic Ca2' with Fura-2-Differentiated cells were washed twice at room temperature with supplemented HBSS containing 1.6 mM CaCL and 0.8 mM MgC12 and resuspended in the same solution at a density of 2-3 X lo6 cells/ml. The suspended cells were loaded with the acetoxymethylester form of Fura-2 (2 p ~ ) and incubated at 37 "C for 40 min. Cells were subsequently washed at room temperature and incubated for an additional 10 min at 37 "C, to allow for further intracellular cleavage of the dye. Fura-2-loaded cells were then washed, resuspended in HBSS (containing the indi- cated Ca" concentrations) at a cell density of 1.5 X lo6 cells/ml, and stored on ice until use. Fura-2 fluorimetry was carried out at 37 "C in quartz cuvettes, as 1.5-ml aliquots of the cell suspensions (3 X lo5 cells/ml) were continuously stirred. Excitation and emission wave- lengths were 339 and 499 nm, respectively. At the end of each measurement, fluorescence signals ( F ) were calibrated to Ca2+ con- centrations by treatment with 50 pg/ml digitonin to lyse the cells (Fmax) followed by addition of 16 mM EGTA (Fmin) (24). Autofluores- cence was measured in cells that were not loaded with the Caz+ indicator; identical values were measured in control and 2 p~ fMLP- treated cells, indicating that this magnitude was Ca2+-independent. Accordingly, the autofluorescence contributions were canceled when calculating [Ca2+]i using the equation: [Ca2+], = Kd(F - Fmin)/(Fmox - F ) (24).

Preparation of Ca2'-EGTA Buffers--Ca"-EGTA buffers for the experiments shown in Fig. 1 were calculated using stability constants included in the FREECA computer program (25). Assay buffers consisted of supplemented HBSS containing 1 mM EGTA, 0.8 mM MgCl,, the indicated concentrations of ATP, and the amounts of CaCl, required to obtain the desired free Ca2+ concentrations. In these experiments, labeled cells were first washed with cold supplemented HBSS without Ca2+ but with 1 mM EGTA, before transference to assay media containing the desired Caz+ concentrations.

Expression ofData-The release of [3H]arachidonate (radioactivity in the supernatant at the end of the incubations) or inositol phos- phates (radioactivity in the column eluates) was corrected by sub- traction of basal release and expressed as percentage of incorporated label. Typical value ranges for incorporated labeled arachidonate and rnyo-inositol were 1.5-2.0 X lo5 and 7-8 X lo5 cpm/assay tube, respectively. Unless otherwise specified, data are representative of results obtained in at least three independent experiments.

RESULTS

Effects of Extracellular Ca2+ on Agonist-stimulated AA Re- lease in HMO Cells: Inhibitory Effect of ATP-The presence of extracellular Ca2+ is a requirement for agonist-stimulated AA release in a variety of cell types (26-28). As shown in Fig. 1, addition of cation to the extracellular medium restored

6604 Dual Regulation of AA Release by Pz Purinoceptors

2 uM IYLP ::.::,” -6 -5 -4 -3 -2

Free Ca“ (log M)

FIG. 1. Dependence of agonist-promoted AA release on ex- tracellular Ca2+ and inhibition of AA release by ATP in EGTA-washed differentiated HL60 cells. Labeled cells were washed for approximately 30 min in Ca2+-free supplemented HBSS containing 1 mM EGTA. This treatment resulted in depletion of Ca2+ from intracellular stores, as determined in a parallel experiment using Fura-2-loaded cells. [3H]AA release was measured in the absence (control) or presence of the indicated agonists at different concentra- tions of extracellular Ca2+ in supplemented HBSS. Values shown in the figure are from single point incubations. Arachidonic acid release was expressed as percentage of the [3H]AA dpm incorporated into the cells. The EC6o values for extracellular Ca2+ in the presence of 2 p M fMLP or 15 pM ATP were 0.26 & 0.05 mM and 0.35 +. 0.05 mM, respectively (mean +. S.D., n = 3). For details see “Experimental Procedures.”

fMLP- and ATP-stimulated AA release in HL60 cells washed for 30 min with Ca2+-free buffers containing 1 mM EGTA (EC50 values for fMLP and ATP were 0.26 f 0.05 and 0.35 f 0.05 mM, respectively). This was expected since both the influx of cation and its intermediate-to-long term mainten- ance in intracellular stores depend on the presence of Ca” in the extracellular medium. Concentrations ranging between 2 and 10 mM extracellular Ca2+ resulted in maximal stimulation of AA release by agonists. In contrast, the omission or addition of extracellular M e (up to 10 mM) did not exert any signif- icant effects on this agonist activity (data not shown). As also seen in Fig. 1, addition of 1 mM ATP resulted in a significant inhibition of fMLP-stimulated AA release which was not abolished by increases in the extracellular Ca2+ concentration. This indicates that the nucleotide effect is not related to Ca2+ chelation. We chose to use 8 mM Ca2+ and 0.8 mM M e as the total extracellular divalent cation concentrations in sub- sequent experiments aimed at the characterization of the nucleotide inhibitory effect. Under these conditions, extracel- lular Ca2+ was saturating for agonist-stimulated AA release at all nucleotide concentrations tested. This cation concen- tration did not affect the viability of HL60 cells as measured by propidium iodide exclusion under fluorescence microscopy. The potencies and efficacies of agonist-promoted AA release were similar at low (1.6 mM) or high (8 mM) Ca2+ (results not shown), in agreement with previous observations (29).

ATP Concentration Responses on Basal and fMLP-stimu- luted Releases of AA or Inositol Phosphates-Concentration- response curves for ATP effects on basal and fMLP-stimu- lated release of AA and inositol phosphates are shown in Fig. 2 ( A and B ) . Compared with the dose-dependent effects of ATP on Ca2+ mobilization and @-glucuronidase secretion in human neutrophils and HL60 cells (3, 5, 6, 8) and on PLD activation in HL60 cells (18), the concentration-response curve for AA release is uniquely biphasic (Fig. 2 4 ) . A concen-

r ‘+2 pM fMLP

- 8 - 7 -6 -5 -4 -3 -2

ATP (log M )

FIG. 2. ATP effects on basal and fMLP-promoted release of AA and inositol phosphates in differentiated HL60 cells: con- centration-response studies. Assays for the release of AA and inositol phosphates were performed using supplemented HBSS con- taining 8 mM CaC12, as described under “Experimental Procedures.” The release of AA (panel A ) was expressed as explained in legend to Fig. 1. Inositol phosphate release (IP2 + IP3) was expressed as the percentage of the total [3H] dpm incorporated into the cells (panel B ) .

tration-dependent stimulatory effect on AA release was de- tected at nucleotide concentrations ranging from 0.3 to 15 p~ ATP (ECBO for stimulation was 3.2 f 0.9 pM); higher concen- trations resulted in a dose-dependent inhibitory effect (ICbo for inhibition was 90 f 11 p ~ ) . The biphasic effect of ATP was also observed on fMLP-stimulated AA release. Low con- centrations of ATP (15 pM) promoted an effect partially additive to that of fMLP; higher concentrations resulted in inhibition of both fMLP- and ATP-dependent stimulations. The EC5,, values for the stimulatory and inhibitory effects of the nucleotide on both basal and fMLP-promoted release were identical, with maximal stimulations occurring in the same range of concentration (10-20 p ~ ) . The nucleotide inhibitory effects were not limited to self- and fMLP-induced release of AA; stimulation mediated by PAF (3-30 PM) was also inhibited up to approximately 60% upon addition of 1 mM ATP (results not shown). In contrast, under the same conditions (8-min incubations) ATP did not inhibit ATP- or fMLP-stimulated release of inositol phosphates (Fig. 2B). ATP showed a concentration-dependent stimulation of ino- sitol phosphate release, which was completely additive to that of fMLP. Similar monophasic concentration-response curves were obtained when measuring the release of inositol phos- phate in shorter incubations (20 s), in the presence or absence of Lick EC50 values for ATP under these conditions were approximately 1 p ~ , either in the presence or absence of fMLP (results not shown).

Relationship between Agonist-promoted Release of AA and Inositol Phosphates-As shown in Fig. 2, the inhibitory effect of ATP on AA release was detected within the concentration range corresponding to maximal stimulation of inositol phos- phate release by the nucleotide. It was therefore possible that the inhibitory effect of ATP on AA release reflected PLC activation by Pz purinergic receptors in these cells (3-6). However, this possibility was ruled out, as other nucleotides which stimulate inositol phosphate release to a level compa-

Dual Regulation of AA Release by P2 Purinoceptors

rable with that of ATP (Fig. 3B) resulted in additive, as opposed to inhibitory, effects on AA release (Fig. 3A).

Effects of ATP on the Permeability of HL60 Celki-Treat- ment of several cell types (30-32) with high concentrations of ATP (e.g. 50-100 p ~ ) can induce permeabilization and leak- age of molecules with molecular mass up to approximately 900 Da. This permeabilizing effect has been attributed to micromolar concentrations of ATP4- in equilibrium with the divalent cation-nucleotide complexes. Fluorescence micros- copy experiments using the fluorescent dye propidium iodide (668 Da) showed that under our experimental conditions (e.g. 8 mM Ca2+, 0.8 mM M$+, and up to 10 mM ATP) no cell permeabilization occurred (data not shown). Even in the complete absence of added divalent cations (and presence of 5 mM each EGTA and EDTA), the optimal conditions for membrane permeabilization by ATP in many cells, 5 mM ATP failed to induce permeabilization of HL60 cells (50 p~ digitonin induced permeabilization of 100% of cells, as a positive control). Therefore, unlike mast cells, macrophage, and transformed fibroblast cells (30-32), but like neutrophils (33) and other cell types (32), HL60 cells are resistant to ATP-dependent membrane permeabilization, presumably be- cause of the lack of a membrane-permeabilizing ATP4- recep- tor.

Effect of ATP on Changes in Intracellular Cu2+-Previous studies have shown stimulation of Ca2+ influx and intracel- lular mobilization by ATP through Pz purinergic receptors in HL60 cells and neutrophils (3, 4, 7, 8). In these experiments low concentrations of ATP (up to 10 p ~ ) were usually em- ployed since they were sufficient to trigger maximal influx and intracellular mobilization of Ca2+. In view of the depend- ence of agonist-promoted AA release on extracellular (Fig. 1) and intracellular Ca2+ (26,28), we studied whether the inhib- itory effects of high concentrations of ATP (230 p ~ ) were due to an impairment in the increase in [Caz+li promoted by agonists. As shown in Fig. 4A, concentrations of ATP asso- ciated with either partial or maximal stimulation (1 and 20 p M , respectively) or with inhibition (1 mM) of AA release all resulted in a maximal increase in [Ca2+]i. The. figure illustrates the overall change in [Ca2+Ii (resulting from the combination of cation mobilization, influx, extrusion, and restorage) in- duced by different ATP concentrations ranging from to

P ’ I 10 A

Control ATP 1 GTP 4 CTP

FIG. 3. Effects of different nucleoside triphosphates on basal and fMLP-promoted release of AA (panel A ) and inositol phosphates (panel B ) in differentiated HL6O cells. The effects of nucleotides (added at 2 mM) were measured in the absence (open bars) or presence (closed bars) of 2 p M fh4LP. Experimental condi- tions were as described for Fig. 2.

A 1.624

f 1.008 .’ 700 1 *: 5 1 5 rr

I t 2 . , 0 - ~

1.680

- . 728

- 5 1.045

- - - : 538 -

3 2 0

- 9 4 1 1

- 0” 252 -

199 L

C

r t

2 ~ 1 0 ~ ’

6605 B

1,089

4 2 1

3 2 8 20 uM ATP

203 250 t J

I I t 2 UM IMLP 2 mM ATP 2 UY IMLP

2 mM ATP

FIG. 4. ATP effects on self- and fMLP-induced increases in [Ca2’Ii in differentiated HL6O cells. [Ca2+], in Fura 2-loaded cells was measured as described under “Experimental Procedures.” Ionic conditions were identical to those described for Fig. 2. Changes in [Ca2+]; were triggered by adding aliquots of the agonist stock solutions (300-fold concentrated) to the cell suspensions. Each transient was obtained with a separate aliquot of the cell suspension. Panel A , short term changes in [Ca’+]; induced by concentrations of ATP (100 nM to 2 mM) associated with the biphasic pattern of AA release. Panel B, comparison of long term changes in [Ca2+Ii induced by 20 PM or 1 mM ATP. The two tracings were graphically superimposed for com- parison. Panels C and D, short and long term changes in [Caz+]; elicited by treatment of cell suspensions with 2 p~ fMLP in the absence or presence of millimolar concentrations of ATP.

2 X M. The changes brought about by Ca2+ mobilization and influx could be easily differentiated by adding appropriate concentrations of LaCb or EGTA which abolish the influx of Ca2+, but leaves Ca2+ mobilization intact (results not shown). The increase in [Ca2+Ii reached maximum at 1 p~ ATP, in agreement with previous studies (3, 4). Following the rapid Ca2+ mobilization (a few seconds), the [Ca2+Ii gradually de- creased over a few minutes; there were no apparent differences in the patterns induced by either 20 p M or 1 mM ATP concentrations (Fig. 423). Furthermore, ATP did not affect the fMLP-stimulated increases in intracellular Ca2+ either (Fig. 4C). In fact, the return of the levels of [Caz+li raised by fMLP to basal was retarded by addition of ATP (Fig. 40). These data indicate that the inhibitory effects of ATP on AA release are not caused by preventing the increase in [Ca2+Ii promoted by agonists.

It is worth mentioning that the different effects of 1 PM ATP on inositol phosphate release (Fig. 2B) and [Ca2+Ii (Fig. 4) are due to differences in the assay times: 8 min and up to approximately 1 min in the experiments reported in these two figures, respectively. Concentration-response studies meas- uring stimulation of inositol phosphate release by ATP fol- lowing 20-9 incubations showed an ECso of approximately 1 p~ with maximal effects obtained at 10-20 p~ ATP, in the presence or absence of fh4LP (results not shown).

Specificity of Nucleotides and Nucleosides in the Inhibition of AA Release-Adenosine effects were examined in view of the inhibition caused by the nucleoside on fMLP-induced superoxide anion production in human neutrophils (34) and glucose production in hepatocytes (35). The inhibitory effects of ATP on AA release in HL60 cells could reflect its hydrolysis to, or contamination by, adenosine. However, the efficacies of

6606 Dual Regulation of AA Release by Pz Purinoceptors

adenosine relative to that of ATP in inhibiting AA release promoted by 2 pM fMLP and 15 p~ ATP were 0.60 k 0.09 (mean f S.D., n = 5) and 0.51 f 0.08 (mean f S.D., n = 5), respectively, although adenosine was more potent (E& = 0.7 f 0.3 phi, n = 5 versus 90 f 11 pM, n = 13, for adenosine and ATP, respectively). Therefore, trace amounts of adenosine, if present, cannot entirely account for the effects of ATP in this system. More importantly, inclusion of adenosine deaminase, which catalyzes the deamination of adenosine into inosine (a nucleoside that does not interact with the adenosine receptor, Ref. 36), completely reversed the effect of adenosine on AA release (Fig, 5B) , but did not alter the biphasic response to ATP (Fig. 5A). Finally, preincubation of differentiated HL60 cells with 1 mM adenosine for 1 h at 37 “C abolishes the ability of adenosine to inhibit the release of AA promoted by 2 PM fMLP or 15 p~ ATP but has no effect on the inhibition of these two parameters by 1 mM ATP (Fig. 6A). These facts argue against the possible involvement of PI purinergic recep- tors in the inhibitory effects elicited by ATP.

Further evidence for the involvement of a P2 purinergic receptor-mediated mechanism in ATP inhibition was ob- tained by studying the effects of different purine and pyrim- idine derivatives (Fig. 7, A and B, respectively). ATP, ATP+, and UTP showed very similar biphasic concentration-re- sponse curves; all other nucleotides tested (ADP, AMP- P(NH)P, AMP-PCP, GTP, XTP, CTP, and TTP) showed only stimulatory effects of lower potency. It is also worth noting that, at maximal inhibitory concentrations, the com- bination of ATP and UTP did not result in additive inhibitory effects (Fig. 7C), suggesting that both nucleotides elicit their effects through a common pathway. As mentioned, concentra- tion-response curves for ADP showed only stimulatory effects with a potency that was approximately 30 times lower than that of ATP (Fig. 7A). Both ADP and AMP were able to inhibit the release of AA stimulated by 15 p~ ATP, although with potencies that were approximately 50 times lower than that of ATP (results not shown). This showed that the inhib- itory effect of ATP cannot be attributed to its conversion into ADP or AMP.

These results are important from several points of view. First, they support the notion of an inhibitory effect mediated by a specific nucleotide receptor, as opposed to a non-specific phenomenon elicited by high concentrations of any nucleo- tide. The UTP response also makes unlikely the possibility that adenosine, by acting through a PI purinergic receptor or

A

lo r B

lo r

-8 -7 -6 -5 -4 -3 -2 cmtr01 15 UY ATP 95 uu ATP

ArP 1109 M) too UH ado

FIG. 5. Effects of adenosine deaminase treatment on the biphasic pattern of AA release induced by ATP. Experimental conditions were as described in the legend to Fig. 2. Mixtures were preincubated at 37 “C in the absence (open circles and bars) or presence (closed circles and bars) of 1 unit/ml adenosine deaminase for 12 min before starting the AA release assay by adding the cell suspension. Control experiment shown in panel B indicates that the enzyme treatment abolishes the inhibitory effect of adenosine (ado) on stimulation of AA release promoted by 15 WM ATP.

15

- 0

c .- - 0 10 s - w v)

2l J W = 5

4 a

ii 0

A

111 IO r

CONTROL 2 uM tMLP uM IMLP +2 mM ATP 2o UM

FIG. 6. Effects of receptor desensitization on regulation of AA release in Bt2cAMP-differentiated HL60 cells. Cells were preincubated in the absence or presence of the specified ligands during the last 60 min of labeling with [3H]AA. Incorporation of [3H]AA in the treated cells did not differ significantly from that in the control group. Cells were then washed as described under “Experimental Procedures” and assayed for AA release under the conditions indi- cated along the x-axis. Data were expressed as mean f range of duplicate determinations. Experiment A, preincubation in the absence or presence of 1 m M adenosine (empty or filled bars, respectively). The efficacy of inhibition of adenosine, relative to that of 1 mM ATP4 on fMLP-promoted AA release in this experiment was 0.77, the highest of the five values averaged (see “Results section; 0.60 f 0.09). Experiment B, preincubation in the absence (empty bars) or presence of 20 WM or 2 mM ATP (filled or hatched bars, respectively).

after its cellular uptake, is responsible for the inhibition of AA release measured at high concentrations of ATP. Second, the similar potencies of the stimulatory and inhibitory effects of ATP, ATP-yS, and UTP on AA release are in agreement with the potencies shown by these nucleotides when interact- ing with PZu purinergic receptors in HL60 cells to activate PLC and increase [Ca”Ii (3-6). This argues in favor of the existence of at least two subtypes of the “PZu” or “nucleotide” receptor expressed in HL60 cells: one involved in stimulation and the other in inhibition of AA release.

Receptor Desensitization Induced by ATP: Time Courses of Stimulatory and Inhibitory Effects on AA Release-Incubation of different cell types with ATP (minutes to hours) results in desensitization of Pz purinergic receptors (37-40). Preincu- bation of neutrophil-like HL60 cells for 1 h at 37 “C with 20 p~ ATP resulted in partial loss of stimulation of AA release by this nucleotide concentration in the subsequent assays, without affecting fMLP-mediated stimulation or the ability of 1 mM ATP to inhibit stimulation by the chemotactic peptide (Fig. 6B). On the other hand, preincubation of cells with 2 mM ATP, while also preserving stimulation of AA release by 2 p~ fMLP, resulted in complete abolishment of

Dual Regulation of AA Rt ?lease by P2 Purinoceptors 6607

other (lower affinity for ligand) to inhibition of AA release. The results shown in Fig. 6B also deny the involvement of

heterologous receptor desensitization in the inhibitory effects of high concentrations of ATP on chemotactic receptor-stim- ulated AA release. This conclusion was further supported by studying the concentration responses of fMLP-stimulated AA release, in the presence or absence of 2 mM ATP. The reduc- tion in the number of fMLP receptors should result in either a decrease in the potency of fMLP for AA release (if the remaining receptors and the amplification that takes place upon occupancy still results in receptor “spareness”) or in the simultaneous reduction in both the efficacy and potency of the agonist (if the reduction in receptor number has proceeded beyond the spareness of the system) (41, 42). Results shown in Fig. 8 indicate that the addition of ATP to the assay mixtures resulted only in a marked reduction in efficacy, without altering the EC50 for fMLP-mediated stimulation of AA. Changes in the apparent affinity of the purinergic recep- tor-ligand interaction could not be studied by a similar ap- proach. Nonetheless, the time courses (Fig. 9) showed that stimulation of AA release by agonists (15 p~ ATP or 2 p M fMLP) takes place during the first 7-10 min of incubation and that 1 mM ATP reduces the initial rate of agonist- promoted AA release, as measured within 5-15 s of reaction. This rapid inhibition is atypical for a desensitization phenom- enon (41). The fact that under the same experimental condi- tions no biphasic effect of ATP on PLC activity in HL60 cells could be detected (Fig. 2B) provides further evidence against homologous receptor desensitization as a basis for the inhi- bition of AA release mediated by ATP. Rather, it supports the idea that the inhibition of AA release by ATP is a direct receptor-mediated transmembrane signaling process.

I t is also worth mentioning that addition of 1 mM ATP 5- 10 min after the start of reaction did not result in any inhibition of AA release promoted by 2 p M fMLP (results not shown). This suggests that the rapid inhibitory effects elicited by the nucleotide are not due to the unlikely enhancement of reuptake/reacylation of [3H]AA released, and diluted isotop- ically (7 x lo5-fold), in the incubation medium.

Involvement of PLA2 in fMLP- and ATP-promoted AA Release in Neutrophil-like HL60 Cells-Results shown in Fig. 10 (A and B ) indicate that the addition of 100 p~ mepacrine, a widely used PLA2 inhibitor, resulted in complete inhibition of agonist-promoted release of arachidonic acid without alter- ation of agonist effects on the production of inositol phos- phates. These results suggest that ATP- or fMLP-promoted

A

-7 -6 -5 -4 -3 -2

-7 -6 .5 -4 -3 -2

or C NUCLEOTIDE (log M)

CONTROL 10 pM UTP +2 mM ATP 2 mM UTP m M ATP 10 pM UTP 2 mM UTP

FIG. 7. Effects of purine and pyrimidine nucleotides on AA

studies. Panel A: ATP (0), ATPrS (O), ADP (A), AMP-P(NH)P release in neutrophil-like HL60 cells: concentration-response

(A), XTP (O), GTP (W), and AMP-PCP (0). Panel B: UTP (V), CTP (+), and TTP (V). Release of AA was expressed as percentage of the maximal stimulation to normalize the results from different experi- ments. Every nucleotide was tested at least in three independent experiments; curves for ATP and UTP represent 13 and 5 independ- ent experiments, respectively. ECm values for stimulation of AA release by ATP and UTP were 3.2 +. 0.9 and 1.2 +. 0.6 p ~ , respectively (mean +. S.D.). ICbo values for the inhibition of self-induced release of AA were 90 & 11 and 89 _C 8 p~ for ATP and UTP, respectively (mean +. S.D.). Panel C, the release of AA (percentage of incorporated) was measured under the conditions described along the x-axis. Assay conditions were as described for Fig. 2.

both stimulation by 20 p~ ATP and inhibition of fMLP- stimulated AA release by 2 mM ATP. This differential pattern of desensitization, elicited by pretreatment with different concentrations of ATP supports the existence of two different receptors: one coupled to stimulation (high affinity) and an-

O t- “ fMLP (loa M)

FIG. 8. Effect of ATP on the concentration response corre- sponding to fMLP-promoted AA release in differentiated HL60 cells. Experiments were carried out with or without inclusion of 2 mM ATP into the assay medium. Experimental conditions were otherwise as described in the legend to Fig. 2.

6608 Dual Regulation of AA Release by P2 Purinoceptors

r A

I 15 uM ATP

Control

d ”A A

4 6 4 10-

2 mM ATP

0 1 5 10 15 20

Time ( m i d FIG. 9. Time course of the inhibitory effects of ATP on self-

and fMLP-promoted release of AA in differentiated HL60 cells. Cell suspensions were prewarmed at 37 “C before addition of 0.005 volumes of vehicle or test substances. Each value was corrected by subtracting the release at time zero (immediately before addition of agonist). Experimental conditions were as described in the legend to Fig. 2.

.. c E MEPACRINE

r o l

h C F A h C F A C F A

FIG. 10. Agonist-induced release of AA and inositol phos- phates in neutrophil-like HL60 cells: effects of mepacrine treatment. Cells were subjected to mepacrine treatment during the last 10 min of the labeling period with [3H]AA. After treatment, cells were washed with HBSS and assayed as described under “Experi- mental Procedures” ( C , control; F, 2 ~ L M WLP; A, 15 #I ATP).

AA release involves PLA, activation in HL60 cells. In addition, comparable levels of phosphatidylcholine,

phosphatidylinositol, or phosphatidylethanolamine were measured after treatment of cells with either 2 WM fMLP, 15 PM ATP, 1 mM ATP, or 2 PM fMLP plus 1 mM ATP (data not shown). This suggested that inhibition of AA release by ATP does not result from a depletion in PLAz substrates caused by the parallel activation of phospholipases C and D by the nucleotide.

DISCUSSION

Recent review articles have discussed the classification of ATP receptors in HL60 cells (10, 11, 43). The consensus indicates that these receptors, which are coupled to PLC, PLA2, and NADPH oxidase activation, show a pattern of agonist selectivity which clearly distinguishes them from other subtypes of Pz purinergic receptors. The order of po- tency of agonists has been the determinant parameter in the classification of ATP receptors, in the absence of selective antagonists. The most distinct pharmacological property of the nucleotide receptors expressed in HL60 cells is the agonist equipotency displayed by UTP and ATP. This led to their recently proposed classification as either a separate subtype of Pz purinergic receptors (‘‘P2U,’’ Ref. 10) or simply as “nu- cleotide” receptors (11), considering the equipotency of a purine and a pyrimidine.

The major finding of the present study is that ATP, ATP? S, and UTP exert a dual regulation of AA release in HL60 cells, compatible with the existence of functionally different sub- types of Pzu receptors: one stimulatory to AA release and displaying a relatively high affinity for agonist, and the other inhibitory and interacting with low affinity. Both stimulation and inhibition were considered mediated by P2u receptors based on the agonist potency orders.

Before establishing this concept we addressed other mech- anisms that could possibly contribute to the inhibition of AA release. In HL60 cells (Fig. 1) as well as in a variety of other cells (26-28), agonist-stimulated AA release is completely dependent on the presence of extracellular Ca2+ and/or in- crease in the cytosolic concentration of this cation. By using appropriate Caz+ concentrations, we clearly showed that the inhibitory effect of ATP was due neither to Ca2+ chelation by ATP (Fig. 1) nor to alterations in the changes in [Ca2+Ii arising from agonist stimulation (Fig. 4).

Although in a few cell types, including mast cells (30), macrophages (31), and transformed fibroblasts (32), high con- centrations of ATP (in equilibrium with micromolar ATP4-) induced cell membrane permeabilization, we failed to detect a cell-permeabilizing effect of ATP in HL60 cells under con- ditions where ATP induced inhibitory effects on AA release, or under conditions optimal for ATP-mediated membrane permeabilization in other cell types. Therefore, HL60 cells, like neutrophils (33) and other cell lines (321, resist nucleo- tide-induced permeabilization. We can then conclude that the inhibitory effect of ATP on AA release in HL60 cells, unlike the nucleotide action on histamine secretion in mast cells (30), is not dependent on cell permeabilization.

Several studies (34) have demonstrated that adenosine, through its interaction with PI purinergic receptors, inhibits agonist-stimulated superoxide anion generation in neutro- phils. A recent study (35) reported a biphasic dose-response curve for ATP-induced glucose production in hepatocytes, with a pattern similar to that reported here for the effect on AA release. This biphasic curve was composed of an ATP stimulatory effect, via Pp purinergic receptors, together with an inhibitory effect on glucose production via uptake of aden-

Dual Regulation of AA Release by P2 Purinoceptors 6609

osine generated by ATP hydrolysis (35). Therefore, the bi- phasic effect of ATP on AA release reported here could be potentially explained by a similar mechanism. In fact, aden- osine (ineffective per se) did have inhibitory effects on both ATP- and fMLP-stimulated AA release, with a potency sig- nificantly higher than that of ATP. However, the following facts indicate that the inhibitory effects of ATP were not linked to adenosine production. First, ATP was more effica- cious than adenosine in its inhibitory effect. Second, the addition of adenosine deaminase did not prevent ATP-me- diated inhibition, but completely abolished adenosine-induced inhibition (Fig. 5 , A and B ) . Third, experiments with UTP, which cannot be metabolized to adenosine, resulted in inhib- itory effects similar to those of ATP (Fig. 7B).

Having discarded the generation of adenosine as responsi- ble for the inhibitory effects detected at high nucleotide concentrations we asked if this inhibition was caused by the direct interaction of ATP with PI receptors. The fact that preincubation of neutrophil-like HL60 cells with adenosine results in desensitization of the inhibitory effects elicited by this ligand, while preserving the inhibition mediated by ATP, argues against this possibility (Fig. 6A).

The concept that the inhibition of agonist-stimulated AA release by ATP, ATPrS, and UTP is a direct Pzu purinergic receptor-mediated transmembrane signaling process is fur- ther supported by the following evidence. l) The inhibitory effects are specific, in that other purine and pyrimidines showed only stimulatory effect on AA release (Fig. 7, A and B ) and ATP did not inhibit PLC activity within the range of concentration tested (Fig. 2B). 2) The unchanged agonist potency (Fig. 8) and the rapid kinetics of inhibition (Fig. 9), together with the absence of inhibitory effect of ATP on both self- and fMLP-mediated activation of PLC (Fig. 2B) , argue against the involvement of receptor desensitization in the inhibitory phenomenon but support a direct, rapid receptor- mediated mechanism. 3) The inhibitory effect of ATP on fMLP-induced AA release could be desensitized by pretreat- ment of cells with 2 mM ATP (Fig. 6B). 4) The order of potency of different purine and pyrimidines, including the similar potencies of ATP, UTP and ATPyS, is absolutely consistent with the pharmacology of the PLC-coupled nucleo- tide receptor expressed in HL60 cells (3,4).

The production of arachidonic acid is required for activation of superoxide production in neutrophils (44). Therefore, our results explain the recent observation that ATP exerts stim- ulatory and inhibitory effects on superoxide production in neutrophils (45). Furthermore, they provide a functional cor- relate to the heterogeneity observed in ATP binding sites, as detected by binding assays in neutrophils (46) and expression cloning in Xenopus oocytes using mRNA extracted from BtzcAMP-differentiated HL60 cells (47). In addition, Green- berg et al. (48) recently demonstrated that low (<IO0 PM) and high (>lo0 PM) concentrations of ATP mediated different patterns of intracellular Ca2+ changes in 5774 macrophages, and they proposed that the different responses were mediated through different receptor subtypes. The pattern of the bi- phasic ATP concentration-response curve here reported is quite reminiscent of the bell-shaped contraction curve elicited by AMP-CPP in guinea pig vas efferens (49). This phenom- enon has been explained by proposing two types of Pz puri- nergic receptors, P2y and PZx, which induce two opposite biological processes: relaxation and contraction of smooth muscle, respectively (1,38). Unlike PPy and Pzx receptors, the two putative “P2u” receptors proposed in this study are cur- rently pharmacologically indistinct and classified only on a functional basis: the release of AA.

I t must be also mentioned that the biphasic ATP concen- tration-response curves observed in our studies are discrepant with a report by Cockcroft and Stutchfield (6), who observed only minimal inhibitory effects at 1 mM ATP. We are unable to find obvious explanations for this discrepancy. A marked difference when comparing the two reports is the higher sensitivity of our assay; the release of AA induced by optimal concentrations of ATP or fMLP reported in the present study (up to 5-10 and 10-15% of incorporated radioactivity, respec- tively) is significantly higher than that measured by Cockcroft and Stutchfield (about 1 and 4%, respectively). However, the inability to detect significant inhibitory effects is not neces- sarily a consequence of the lower responses detected in the assay; in our hands, the absence of unlabeled arachidonic acid in the assay medium, which resulted in considerably lower levels of [3H]AA released, was not an obstacle to detect the nucleotide inhibitory effects (Fig. 11).

The high efficacies of agonist-induced AA release in our assay are due to the presence of unlabeled AA (0.2 mM) in the reaction medium. Omission of unlabeled AA dramatically decreased the amount of [3H]AA released in the medium upon cell stimulation (Fig. 11). In neutrophils, the reacylation of released AA into lysophospholipids is very rapid (50), and accordingly, quantitative measurement of the changes in en- dogenous lysophospholipids as a function of PLA, activity has not been possible (results not shown and Ref. 22). In this way, the excess of unlabeled AA in the medium is used as substrate for the reacylation reactions, allowing the accumu- lation of the [3H]AA released by agonists. A similar effect of unlabeled AA was observed when measuring the release of [3H]AA from labeled hepatocytes (51).

The biphasic regulation of AA release by ATP and UTP may have physiopathological relevance. Platelets contain sig- nificant amounts of adenine and uracil nucleotides in their dense granules, which are released upon cell activation. The interaction between platelets and neutrophils has been docu- mented following tissue injury and in a number of noninfec- tious inflammatory diseases (52 ) . Thrombin-mediated acti- vation of platelet suspensions ranging from lo6 to 10’ cells/ ml results in the release of 6-800 p M ATP (45), a concentra- tion interval which was associated with clearly different re- sponses in our experiments. It is then conceivable that the ability of neutrophils to stimulate or inhibit the release of AA and its metabolites in response to varying concentrations of ATP might constitute a mechanism to propagate or limit

25 i W v) a W

10 U a I .= - 5

0

7 p 1 5 u M A T P

/ / I 1 mH ATP 2 UM tMLP

1 mM ATP

CTRL.

-6 -7 -6 -5 -4 -3

Unlabeled A A in assay medium (log M)

FIG. 11. The release of [’HIAA promoted by agonists in suspensions of neutrophil-like HL60 cells depends on the con- centration of unlabeled arachidonate added to the extracel- lular medium. Experimental conditions were otherwise as described in the legend to Fig. 2.

6610 Dual Regulation of AA Release by P2 Purinoceptors

inflammation, respectively, as part of the network of inter- actions among inflammatory cell neighbors.

The intracellular mechanism underlying the dual regulation of AA release by P2” purinergic receptors here described is still unknown to us. However, AA released upon ATP or fMLP stimulation is produced by a PLA2-catalyzed reaction (Fig. 10). Both the fMLP receptor and the stimulatory type of P2u receptor seem directly coupled to the activation of PLA, via a pertussis toxin-sensitive G-protein (6). The inhib- itory subtype of this P2u purinergic receptor may negatively regulate any step in the signaling pathway between stimula- tory receptors and PLA,, through an indirect intracellular pathway. It is possible that the inhibition of AA release elicited by occupancy of the low affinity PZu subtype expressed in HL60 cells might be secondary to changes in the levels of CAMP. Evidence for this linkage, although circumstantial, derives from the inhibitory effects of agents that increase cAMP levels (such as PGE, and forskolin) on the release of AA promoted by 2 ~ L M fMLP, 15 p~ ATP, or 15 p~ UTP in Bt2cAMP-differentiated cells (results not shown), in agree- ment with previously reported effects of PGE, on fMLP- induced AA release, superoxide anion formation, and inositol phospholipid metabolism in guinea pig neutrophils (53). Ar- guing against this possibility is the finding that high concen- trations of ATP and UTP do not produce any significant changes in cAMP content in differentiated HL60 cells.3 Al- ternatively, an inhibitory G-protein might directly couple the inhibitory subtype of the Pzu purinergic receptor to the inhi- bition of PLA, in HL60 cells, as ubiquitously found for adenylyl cyclase and recently demonstrated for PLC in FRTL5 cells (54). This possibility is supported by the finding that expression of a chimeric G./Gi2a subunit results in inhi- bition of purinergic receptor activation of PLA, in Chinese hamster ovary cells (16). These possible mechanisms are currently being investigated in our laboratory.

Acknowledgments-We are very grateful to G. Dubyak and M. Kester for discussions and advice and to G. Bright for assistance in the fluorescence microscopy studies.

REFERENCES

1. Gordon, J. L. (1986) Biochem. J. 223 , 309-319 2. Burnstock, G. (1978) in Cell Membrane Receptors for Drugs and

Hormones (Straub, R. W., and Bulis, L., eds) pp. 107-118, Raven Press, New York

3. Dubyak, G. R., Cowen, D. S., and Mueller, L. M. (1988) J. Biol. Chem. 263,18108-18117

4. Cowen, D. S., Lazarus, H. M., Shurin, S. B., Stoll, S. E., and Dubyak, G. R. (1989) J. Clin. Znuest. 8 3 , 1651-1660

5. Cockcroft, S., and Stutchfield, J . (1989) FEBS Lett. 245,25-29 6. Cockcroft, S., and Stutchfield, J. (1989) Biochem. J. 263, 715-

7. Kuhns, D. B., Wright, D. G., Natk, J., Kaplan, S. S., and Basford,

8. Kuroki, M., Takeshige, K., and Minakami, S. (1989) Biochim.

9. Cowen, D. S., Sanders, M., and Dubyak, G. (1990) Biochim.

723

R. E. (1988) Lab. Znuest. 58,448-453

Biophys. Acta 1012,103-106

Biophys. Acta 1053 , 195-203 10. Dubyak, G. R. (1991) Am. J. Respir. Cell Mol. Bwl. 4 , 295-300 11. O’Connor, S. E., Dainty, I. A., and Leff, P. (1991) Trends Phar-

12. Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K.

13. Charest, R., Blackmore, P. F., and Exton, J. H. (1985) J. Biol.

14. Okajima, F., Tokumitsu, Y., Kondo, Y., and Ui, M. (1987) J. Biol.

15. Bruner, G., and Murphy, S. (1990) J. Neurochem. 55,1569-1575

M. Xing, T. Blaha, and R. Mattera, unpublished observations.

macol. Sci. 12, 137-141

(1990) J. Bid. Chem. 265 , 13501-13507

Chem. 260 , 15789-15794

Chem. 262,13483-13490

16. Gupta, S., Diez, E., Heasley, L. E., Osawa, S., and Johnson, G.

17. Bocchino, S. B. , Blackmore, P. F., Wilson, P. B., and Exton, J.

18. Xie, M., and Dubyak, G. R. (1991), Biochem. J. 278,81-89 19. Okajima, F., and Kondo, Y. (1990) J. Bwl. Chem. 266 , 21741-

20. Griffin, H. D., and Hawthorne, J. N. (1978) Biochem. J. 176,

21. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol.

22. Walsh, C. E., Waite, B. M., Thomas, M. J., and Dechatelet, L. R.

23. Yoss, E. B., Spannhake, E. W., Flynn, J. T., Fish, J. E., and

24. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol.

25. Fabiato, A. (1979) J. Physiol. 75, 463-505 26. Brooks, R. C., McCarthy, K. D., Lapetina, E. G., and Morell, P.

(1989) J. Biol. Chem. 2 6 4 , 20147-20153 27. Slivka, S. R., and Insel, P. A. (1988) J. Biol. Chem. 263 , 14640-

14647 28. Nakashima, S., Suganuma, A., Matsui, A., Hattori, H., Sato, M.,

Takenaka, A., and Nozawa, Y. (1989) Biochem. J. 259 , 139- 144

L. (1990) Science 249, 662-666

H. (1987) J. Biol. Chem. 262,15309-15315

21748

541-552

37,911-917

(1981) J. Biol. Chem. 256, 7228-7234

Peters (1990) Am. J. Respir. Cell Mol. Biol. 2 , 69-80

Chem. 260,3440-3450

29. Bokoch, G., and Gilman, A. G. (1984) Cell 39, 301-308 30. Cockcroft, S., and Gomperts, B. D. (1979) J. Physiol. 2 9 6 , 229-

31. Steinberg, T. H., Newman, A. S., Swanson, J. A., and Silverstein,

32. Heppel, L. A,, Weisman, G. A., and Friedberg, I. (1985) J. Membr.

33. Seifert, R., Wenzel, K., Eckstein, F., and Schultz, G. (1989) Eur.

34. Cronstein, B. N., Daguma, L., Nichols, D., Hutchison, A. J., and

35. Oetgen, E., Schweickhardt, C. Unthan-Fechner, K., and Probst,

36. Wolff. J.. Londos, C.. Cook. G. H. (1978) Arch. Biochem. Bio~hvs.

243

S. C. (1987) J. Biol. Chem. 262,8884-8888

Biol. 8 6 , 189-196

J. Biochem. 181 , 277-285

Williams, M. (1989) J. Clin. Znuest. 85, 1150-1157

I. (1990) Biochem. J. 2 7 1 , 337-344 . .

1 9 i , 161-168 _ -

37. Gonzalez. F. A,. Bonauace. E.. Belzer. I.. Friedbere. I.. and HeDoel. L. A. (1989) biocheh. Biophys. Res. Commun.-164, 706-ff3 ’

38. Burnstock, G., and Kennedy, C. (1985) Gen. Phurmacol. 16,433- 440

39. Gonzalez, F. A., Heppel, L. A., Gross, D. J., Webb, W. W., and Parries, G . (1988) Biochem. Biophys. Res. Commun. 151,1205- 1212

40. Martin, M. W., and Harden, T. K. (1989) J. Biol. Chem. 264, 19535-19539

41. Harden, T. K. (1983) Phurmacol Reu. 35, 5-32 42. Kenakin, T. P., and Morgan, P. H. (1989) Mol. Phurmacol. 35 ,

43. Seifert, R., and Schultz, G. (1989) Trends Phurmacol. Sci. 10,

44. Henderson, L. M., Chappell, J. B., and Jones, 0. T. G. (1989)

45. Naum, C. C., Kaplan, S. S., and Basford, R. E. (1991) J. Leukocyte

46. Tarapchak, S., Yu, G., and Ward, P. (1991) FASEB J. 5 , 5695

47. Murphy, P. M., and Tiffany, H. L. (1990) J. Biol. Chem. 265,

48. Greenberg, S., Di Virgilio, F., Steinberg, T. H., and Silverstein, S. C. (1988) J. Biol. Chem. 263,10337-10343

49. Fedan, J. S., Hogaboom, G. K., Wesfall, D. P., and O’Donnell, J. P. (1982) Eur. J. Pharmacol. 81 , 193-204

50. Rubin, R. P., Sink, L. E., Schrey, M. P., Day, A. R., Liao, C. S., and Freer, R. J. (1979) Biochem. Biophys. Res. Commun. 90,

51. Glende, E. A.. Jr., and Pushpendran, C. K. (1986) Biochem. Phurmacol. 35,3301-3307

52. Weksler, B. (1988) in Inflammation: Basic Principles and Clinical Correlates (Gallin, J. I., Goldstein, I. M., and Snyderman, R., eds) pp. 543-557, Raven Press, New York

53. Takenawa, T., Ishitoya, J., and Nagai, Y. (1986) J. Biol. Chem. 261 , 1092-1098)

54. Bizzarri, C., Girolamo, M. D., D’Orazio, M. C., and Corda, D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4889-4893

214-222

365-369

Biochem. J. 264, 249-255

Bid 49,83-89

(Abstr.)

11615-11621

1364-1370