Pertussis Toxin Produces Differential Inhibitory Effects on Basal, Pa-purinergic, and Chemotactic Peptide-stimulated Inositol Phospholipid Breakdown in HL-60 Cells and HL-60 Cell Membranes*

P2-purinergic receptor agonists (UTP) and formylated peptide receptor agonists (FMLP) were found to be equally efficacious in eliciting rapid 6-7-fold increases in inositol polyphosphate accumulation in differentiated HL-60 granulocytes. The activation of this response by either agonist was substantially but incompletely inhibited in cells treated with pertussis toxin. Thus, in cells containing only 1-10% of the control level of non-ADP-ribosylated Gi-2/3, UTP induced rapid 2-fold increases in inositol polyphosphate accumulation whereas smaller 50% increases were observed in FMLP-stimulated cells. Washed membranes prepared from control and toxin-treated HL-60 cells were used to characterize this toxin-insensitive activation of phospholipase C further. The agonist-independent stimulation of phospholipase C by either millimolar Ca2+ or the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was only modestly attenuated by toxin treatment. There was a 70-80% decrease in the rate and extent of phospholipase C activity stimulated by GTP per se in the absence of receptor agonists. The rate and extent of FMLP-induced potentiation of GTP-dependent phospholipase C activity were also inhibited by greater than 80% in toxin-treated membranes. Conversely, the potency and efficacy characterizing UTP-induced potentiation of GTP-dependent phospholipase C activity were only modestly attenuated (less than 20% inhibition). The results indicate that P2-purinergic receptors (and perhaps other Ca2(+)-mobilizing receptors) activate both pertussis toxin-sensitive and toxin-insensitive pathways for phospholipase C regulation in phagocytic leukocytes.

The activation of this response by either agonist was substantially but incompletely inhibited in cells treated with pertussis toxin. Thus, in cells containing only l-10% of the control level of non-ADP-ribosylated Gi-z/s, UTP induced rapid a-fold increases in inositol polyphosphate accumulation whereas smaller 50% increases were observed in FMLP-stimulated cells. Washed membranes prepared from control and toxin-treated HL-60 cells were used to characterize this toxin-insensitive activation of phospholipase C further. The agonist-independent stimulation of phospholipase C by either millimolar Ca2' or the nonhydrolyzable GTP analog guanosine 5'-0-(3-thiotriphosphate) (GTPrS) was only modestly attenuated by toxin treatment.
There was a 70-80% decrease in the rate and extent of phospholipase C activity stimulated by GTPper se in the absence of receptor agonists.
The rate and extent of FMLPinduced potentiation of GTP-dependent phospholipase C activity were also inhibited by >80% in toxin-treated membranes.
Conversely, the potency and efficacy characterizing UTP-induced potentiation of GTP-dependent phospholipase C activity were only modestly attentuated (<20% inhibition).
The results indicate that Pa-purinergic receptors (and perhaps other Ca2+mobilizing receptors) activate both pertussis toxin-sensitive and toxin-insensitive pathways for phospholipase C regulation in phagocytic leukocytes.
The ability of pertussis toxin to inhibit formylated peptide receptor-activated GTP-dependent inositol phospholipid breakdown in phagocytic leukocytes such as neutrophils and differentiated HL-60 cells has been studied and described extensively (l-4). Considerable effort has been directed toward characterizing the regulation of phospholipase C effector enzymes by those G-proteins that are: 1) known to be expressed by phagocytic leukocytes, and 2) are substrates for pertussis-catalyzed ADP-ribosylation. Polakis et al. (5)  reported that the FMLP' receptor co-purifies with a 40-kDa GTP-binding protein that is the major pertussis toxin substrate in neutrophils and HL-60 cells (5). Recent immunochemical and molecular cloning studies from several laboratories have identified this protein as the ai.l-subtype from the G, "superfamily" of regulatory proteins (6,7). These and other studies have demonstrated that neutrophils and HL-60 cells also express Gi.3 but no readily detectable amounts of Gi.1 or G,. Thus, Gi.2 and Gi.3 appear to be the only pertussis toxinsensitive G-proteins expressed in HL-60 cells. Recent studies by Gierschik et al. (8) have indicated that the FMLP receptor can interact functionally with G;.x, in addition to Gi.2, in membranes from differentiated HL-60 cells. Despite the considerable evidence indicating that formylated peptide receptors are coupled to Gi.z/s, important questions concerning the role of specific G-proteins in the regulation of GTP-dependent inositol phospholipid breakdown in phagocytic leukocytes remain unresolved. Kikuchi et al. (9) reported that both rat brain Gi (isotype unspecified) and G, could reconstitute FMLP receptor-phospholipase C coupling when added to HL-60 granulocyte membranes wherein the native Gi-type proteins were ADP-ribosylated by pertussis toxin. However, there is no direct evidence as to whether native Gi.2 or Gi.3 (or both) mediates activation of the inositolphospholipid specific phospholipase C isozyme(s) expressed in these cells. Moreover, the mechanism(s) whereby pertussis toxin interferes with agonist-induced activation of the phospholipase is only partially understood. Although pertussis toxin treatment is known to uncouple G,.* from agonistoccupied FMLP receptors significantly (3), it has not been ascertained whether interaction of the G-protein with unoccupied receptors, guanine nucleotides, or the phospholipase effector may also be attenuated by ADP-ribosylation.
Finally were preincubated for 5 min at 37 "C prior to the addition of agonists.
After 15 s, the reactions were terminated, and the samples were processed for analysis of IP, and IP3 content as described above.

In Vitro Ribosylation
Studies-Membranes were isolated from control or pertussis toxin-treated cells as described above.
In some experiments, the cells were pretreated with 3 mM diisopropylfluorophosphate for 20 min at 4 "C! prior to lysis.

Effects of Pertussis Toxin Treatment on P2-purinergic
Versus FMLP-stimulated Inositol Polyphosphate Formation in Intact HL-60 Cells-UTP is an agonist with a potency and efficacy equal to that of ATP in stimulating inositol phospholipid hydrolysis and Ca2+ mobilization in human neutrophil

Pertussis
Toxin Effects on GTP-dependent Phospholipase C Activity and monocyte cell types (12,14). As an agonist for in uitro studies of Pp-purinergic receptor effects on the phosphatidylinositol phospholipase C, UTP has the advantage of being a poor substrate for membrane-associated lipid kinases (14). As shown in Fig. 1, maximally activating concentrations of UTP or FMLP were equally efficacious in stimulating rapid accumulation of IP, (4-fold increases within 15 s) and IP, (6-fold increases in 15 s) in intact HL-60 granulocytes. The EC& for UTP stimulation was approximately 3 nM whereas that for FMLP was 30 nM. Treatment of HL-60 granulocytes with high concentrations of pertussis toxin for 3 h significantly reduced the efficacies of both FMLP and UTP in stimulating inositol polyphosphate accumulation. However, the maximal extent of the pertussis toxin-induced inhibition was invariably smaller when UTP (or ATP) rather than FMLP was the agonist. This differential sensitivity of P2-purinergic receptors versus FMLP receptors to inhibition by pertussis toxin was very reproducible. It is important to note that varying the concentration of pertussis toxin (250-1000 rig/ml) used to treat the cells during the 3-h incubation had little effect on the magnitude of inhibition of the response to FMLP or UTP. Cells treated with this range of toxin concentrations were characterized by a 91 f 1% (mean f S.E., n = 7) inhibition of maximal FMLP-induced inositol polyphosphate accumulation. In these same cells there was an average 78 f 2% (n = 8) inhibition of the maximal UTP-induced response.

Quantitation of Pertussis Toxin Substrates in Membranes
Isolated from HL-60 Granulocytes-Given the previous results, it was important to determine the residual content of unribosylated Gi.2,3 in HL granulocytes treated with pertussis toxin under a variety of incubation conditions. However, precise quantitation of the residual substrates for in vitro toxin-catalyzed ADP-ribosylation requires that the activated toxin have unimpaired access to the relevant membraneassociated protein substrates. Membranes are routinely prepared from neutrophils and HL-60 cells lysed by nitrogen cavitation (4,5,14,19). The plasma membranes of cells lysed by this method re-form vesicles that may not be permeated readily by the catalytically active 26-kDa A subunit of pertus- sis toxin. Consistent with this possibility, we found that the rate and extent of toxin-catalyzed incorporation of [32P]ADPribose into the major 40-41-kDa substrate of freshly isolated HL-60 membranes are increased by a greater than IO-fold factor if certain detergents such as Thesit (Lubrol PX and digitonin are also effective, data not shown) are included in the in vitro ADP-ribosylation reaction medium (Fig. 2). This is in agreement with the studies of Ribeiro-Neto et al. (22) who reported that Lubrol increased pertussis toxin-catalyzed ADP-ribosylation of thyroid membranes. These investigators also noted that inclusion of detergent produced a 2-3-fold potentiation of the ADP-ribosylation of purified G, (derived from human erythrocytes); this latter result suggests that detergent may additionally affect the conformational states of the toxin and/or the G-protein substrate. Although the use of detergents increased accessibility of the HL-60 membrane G-proteins to the toxin, it also increased the accessibility to endogenous proteases that are particularly abundant in the crude membrane preparations used in the present studies (Fig. 2). In the presence of detergent, there was a very rapid incorporation of ADP-ribose into the major 40-41-kDa substrate after an initial 2-min lag. This reaction approached >75% completion within 5 min (Fig. 2B); there was only a minor 5-10% increase when incubations were extended from 10 to 15 min (data not shown). However, with incubation times longer than 10 min, three additional 32P-labeled bands appeared; these had molecular masses of 30, 27, and Cl0 kDa (migration with dye front). With longer incubation times, there was a progressive loss of radioactivity in the major 40-41-kDa substrate which was quantitatively matched by increased 32P counts associated with the lower molecular mass species (Fig. 2B). The rate of this apparent proteolysis of the 40-41-kDa substrate could be reduced but not eliminated by 1) preincubation of the cells with diisopropylfluorophosphate prior to lysis; and 2) inclusion of divalent cation chelators and leupeptin in the reaction medium. Presumably, this proteolysis might also be reduced by separating the proteasecontaining azurophilic granules from the plasma membrane fraction by gradient centrifugation (19); however, this is not practical when dealing with the relatively low numbers of cells pretreated with pertussis toxin. Thus, we routinely employed lo-min incubations in the presence of 0.05% Thesit to ensure maximal incorporation of [32P]ADP-ribose into the 40-41-kDa substrate (combined ai. and ai.3) but minimal proteolytic degradation of these G-protein a-subunits.
Correlation between ADP-ribosylation of Gi.2,3 and Toxin Effects on UTP-and FMLP-stimulated Inositol Polyphosphate Accumulation-Additional experiments were designed to correlate the apparent pertussis toxin-resistant component of agonist-induced phospholipase C activity with the residual content of nonribosylated Gi.2,3. Parallel cultures of unlabeled and [3H]inositol-labeled HL-60 granulocytes were incubated with various concentrations of toxin for 3 or 15 h. Membranes prepared from the unlabeled cells were assayed for unmodified Gi.2/3 content using the in vitro ADP-ribosylation protocol described above (Fig. 3 Membranes isolated from HL-60 granulocytes were used as substrate for pertussis toxincatalyzed ADP-ribosylation as described under "Experimental Procedures." ADP-ribosylation was assayed in standard reaction medium in either the absence (-) or presence (+) of 0.05% Thesit detergent. Reactions were initiated by the addition of membranes (10 pg of protein/assay) and were stopped at the indicated times by the addition of trichloroacetic acid. Parallel samples of HL-60 granulocytes were pretreated with the indicated concentrations of pertussis toxin for 3 or 15 h. Membranes isolated from these cells were used as substrate for pertussis toxin-catalyzed ADP-ribosylation of Gi.213 as described under "Experimental Procedures" and in the Fig. 2 legend. ADP-ribosylation was assayed in standard reaction medium containing 0.05% Thesit detergent. Reactions were initiated by the addition of membranes (15 rg of protein/assay) and were stopped after 10 min by the addition of trichloroacetic acid. Panel A shows the autoradiograms from two separate experiments; panel B details average + range of the relative (control = 100%) "'P cpm incorporated into the 40-41-kDa substrate. decrease in unribosylated G-protein observed in cells treated with toxin for prolonged times was not matched by an additional inhibition of agonist-induced inositol polyphosphate accumulation (Fig. 4)  Membranes isolated from ["Hlinositollabeled HL-60 granulocytes were incubated for the indicated times at 37 "C in basic assay medium (described under "Experimental Procedures") containing 350 nM free Ca2+ (first panel from left). Incubation media additionally contained 1 FM GTP (second panel), 1 @M GTP plus 30 pM UTP (thirdpanel), or 1 pM GTP plus 10 FM FMLP (fourth panel). Circles (0) represent data from membranes of control cells; squares (m) represent data from membranes of cells treated for 3 h with 500 rig/ml pertussis toxin. Accumulation of ["Hlinositol polyphosphates was quantified as described under "Experimental Procedures." Data points represent the average of duplicate determinations from a single experiment. These data are representative of six separate experiments. 4,9). This was done to offset possible reduction in the levels of "H-polyphosphoinositides through the action of phosphomonoesterases.
Since P,-purinergic receptors were a focus of the present study, nucleotides were included only as specified. Figs. 5 and 6 and Table I Membranes isolated from ["Hlinositol-labeled HL-60 granulocytes were incubated for 2 min at 37 "C in basic assay medium  Table I). The inclusion of either 30 PM UTP or 10 PM FMLP produced an additional 1.5-2.5-fold increase in the rate of this GTPdependent phospholipase C activity. We have verified previously that this effect of UTP (or other Pp-purinergic agonists) is not due to elevation of membrane polyphosphoinositide levels or to inhibition of GTP catabolism by membraneassociatedphosphatases/nucleotidases (14). The threshold for UTP action was approximately 100 nM, the EC&) was about 1.8 PM, and near maximal activation was obtained with 30-100 PM. These values are similar to those characterizing the effects of UTP on intact cells (Fig. 1). Conversely, the FMLP dose-response relationship in control membranes was markedly different from that observed in intact cells. The threshold was 30 nM whereas the ECso was at least 300 nM; a plateau in activation was not observed even with 10 yM FMLP. The data presented in Fig. 6 and Table I also demonstrate that efficacy of FMLP (mean 1.8-fold stimulation) was invariably less than that of UTP (mean 2. 5-fold stimulation) in this in uitro phospholipase C assay system. Parallel studies performed on membranes isolated from cells pretreated with pertussis toxin revealed several differences. First, as shown in Figs. 5 and 6 and Table I, the "basal" rate of GTP-dependent inositol polyphosphate formation (i.e. measured in the absence of receptor agonists) was significantly inhibited (mean inhibition = 72%) in the toxin-treated membranes.
It should be emphasized that pertussis toxin treatment did not alter the levels of the various membrane ["Hlinositol phospholipids (data not shown); nor did it significantly alter activation of the membrane-associated phospho- C activity in membranes from HL-60 granulocytes ["H]Inositol-labeled differentiated HL-60 cells were incubated in the absence (control) or presence (PTX) of 500-2000 &ml pertussis toxin for 3 h. Membranes isolated from these cells were then incubated for 1-2 min at 37 "C in thebasic assay medium (described under "Experimental Procedures") containing either 350 nM free Ca2+ or 1 mM free Ca". Parallel samples also contained (in addition to 350 nM free Ca'+): no agonists (None), 1 FM GTP, 1 fiM GTP plus 30 PM UTP, or 1 PM GTP plus 10 PM FMLP. Accumulation of [3H]inositol polyphosphates was quantified as described under "Experimental Procedures." In each experiment, every incubation condition was assayed in duplicate. Listed values represent the mean + standard error of data from six separate experiments. Percentage changes in total activity are defined as 1 -((agonist -none)rrx/(agonist -none),,,&. Percentage changes in GTP-dependent activity are defined as   (Table I). As reported in previous studies (l-3, 9), the ability of FMLP to potentiate GTPdependent inositol polyphosphate accumulation was markedly reduced in membranes prepared from cells that had been pretreated with pertussis toxin (Figs. 5 and 6 and Table I). Time course studies (Fig. 5) revealed that both the total extent of (FMLP + GTP)-stimulated inositol polyphosphate accumulation and the differential ((FMLP + GTP) -GTP) rate of accumulation were markedly depressed in the membranes from the toxin-treated cells. In contrast, although the total extent of UTP-stimulated GTP-dependent inositol polyphosphate accumulation was modestly depressed in the same toxin-treated membranes, the differential ((UTP + GTP) -GTP) rate of agonist-induced accumulation was not inhibited significantly (Fig. 5). The dose-response relationships (Fig. 6) characterizing the effects of FMLP and UTP underscored further the differential abilities of these agonists to potentiate GTP-dependent phospholipase C activity in membranes from toxin-treated cells. Both the potency and efficacy of FMLP were significantly reduced (Fig. 6B). Conversely, the UTP dose-response curve for the toxin-treated membranes paralleled that for the control membranes (Fig. 6A). The threshold (<300 nM UTP) and ECso (approximately 1.8 PM UTP) for UTP stimulation of GTP-dependent activity were identical in membranes prepared from control or pertussis toxintreated cells; the efficacy of UTP stimulation was reduced by only X-20% in the toxin-treated membranes. As summarized in Table I, toxin-treated membranes were characterized by a mean 72% decrease in the GTP-dependent activity measured in the absence of receptor agonists. Likewise, there was a mean 71% decrease in the total FMLP + GTP-stimulated inositol polyphosphate accumulation. Conversely, the total phospholipase C activity stimulated by UTP + GTP was inhibited by only 36%. This difference in the inhibition of inositol polyphosphate release elicited by the two receptor agonists was even more striking when the differential ((agonist + GTP) -GTP) activities were compared. The additional GTP-dependent activity observed in the presence of UTP was inhibited by only 14% compared with the 82% reduction in FMLP-induced action. The above data were obtained using membranes prepared from cells treated for 3-4 h with ~500 rig/ml pertussis toxin. As noted previously, such membranes retain only lo-15% of the normal content of nonribosylated Gi.213. It is possible, however, that in the presence of high numbers of occupied Pz-purinergic receptors, this residual amount of unmodified G-protein was sufficient to mediate the appreciable stimulation of GTP-dependent phospholipase C observed in the membrane experiments.
To test this possibility, similar experiments were performed using membranes isolated from cells treated with pertussis toxin for 15 h. In the experiment illustrated in Fig. 7B, such membranes contained only 1% of the normal content of unmodified Gi.213 as compared with 11% content measured in a membranes derived from a parallel culture of cells treated for 4 h (Fig. 7A). Despite the differences in unmodified G,.2,3 content, essentially similar patterns of altered phospholipase C regulation were observed. In both sets of toxin-treated membranes, the FMLP-induced potentiation of GTP-dependent activity was reduced by 65-70% whereas the potentiation elicited by UTP was decreased by only lo-12%.
In both types of toxin-treated membranes, the GTP-dependent activity measured in the absence of receptor agonists was decreased by 50-65%. Likewise, the Ca2+-stimulated phospholipase C activities measured in both sets of toxin-treated membranes were not significantly different from those observed in control membranes.
The only functional difference noted in membranes from cells treated with toxin for prolonged times was a 38 + 7% (n = 4) reduction in the maximal GTPyS-stimulated phospholipase C activity; a much smaller 12 f 7% inhibition (n = 5) in this parameter was observed when membranes were prepared from cells treated with toxin for 3-4 h. This latter observation suggests that prolonged exposure to pertussis toxin may down-regulate the total amount of G-protein(s) that is functionally coupled to phospholipase C effector enzymes. Additive Stimulation of GTP-dependent Phospholipase C Activity in HL-60 Membranes by Pp-purinergic Agonists and Formylated Peptides-As described in the previous experiments, membranes prepared from pertussis toxin-pretreated cells exhibited significantly (70%) reduced rates of GTPdependent inositol polyphosphate release in the absence of receptor agonists. Other investigators have reported that basal GTP-dependent activation of phospholipase C is unaltered in membranes prepared from neutrophils (2,3) and differentiated HL-60 cells pretreated with pertussis toxin (1,4,9). However, the membranes used in these studies were prepared and stored in the presence of millimolar ATP. Thus, the subsequently assayed phospholipase activity was measured in the presence of 0.05-l mM ATP. Under these conditions, the basal GTP-stimulated inositol polyphosphate formation re- Gi.z,s. Parallel samples of unlabeled and ["Hlinositol-labeled HL-60 granulocytes were pretreated with 1 pg/ml pertussis toxin for either 4 h (panel A) or 15 h (panel B). Membranes were prepared as described previously. Unlabeled membranes were then assayed in uitro for pertussis toxin-catalyzed ["'PIADP-ribosylation of Gi.2,:1 (40-41-kDa substrate) as described in Fig. 3. Lanes I, 2, and 3 of each autoradiogram illustrate "P incorporation in control membranes measured at 5, 10, and 20 min, respectively. Lanes 4, 5, and 6 represent the corresponding time points in membranes isolated from toxintreated cells. "H-Labeled membranes were assayed for inositol polyphosphate release during 1-min incubations (37 "C) in basic assay medium (350 nM free Ca") containing either 1 pM GTP, 1 pM GTP plus 30 PM UTP, 1 PM GTP plus 10 pM FMLP, 10 ~.IM GTPrS, or 1 mM free Ca'+. Open bars (0) represent data from control membranes; cross-hatched bars (m) represent data from pertussis toxin-treated membranes. Inositol polyphosphate accumulation has been normalized relative to the total content of ["Hlinositol phospholipid in each membrane preparation. Data points show the average + range of duplicate measurements from single experiments. Each experiment is representative of results obtained from two separate experiments. ported in such studies was actually a measurement of (GTP + ATP)-stimulated activity due to the unintentional occupation of Pz-purinergic receptors. Similarly, the measurements of (FMLP + GTP)-induced activity were actually measurements of stimulation triggered by (FMLP + GTP + ATP). This is significant given the relative pertussis toxin insensitivity of P,-purinergic-induced phospholipase C activation observed in the isolated membranes. As shown in Table II,  simultaneous exposure of membranes to both FMLP and P,purinergic agonists (either 1 mM ATP or 30 PM UTP) induced an additive increase in the rate and extent of agonist-stimulated GTP-dependent phospholipase C activity. Although pertussis toxin inhibited the FMLP-induced component of this additive inositol polyphosphate accumulation, there was relatively little inhibition of the ATP-or UTP-induced component. When ATP was included as part of a basic incubation medium, the nominally basal GTP-induced activation of phospholipase C was relatively unchanged in membranes isolated from cells treated with pertussis toxin. This same pattern of differential receptor-stimulated phospholipase C activation was observed in toxin-treated membranes wherein residual content of unmodified G, 2,:1 ranged from 1 to 15% of control values. Thus, the routine inclusion of ATP in in vitro studies of G-protein-regulated phospholipase activity can significantly complicate the interpretation of data if the membranes contain receptors for ATP. This may have widespread significance given the growing number of cell types in which extracellular ATP has been reported to activate transmembrane signaling processes known (or hypothesized) to involve mediation by GTP-binding regulatory proteins.

DISCUSSION
These studies provide several observations pertinent to understanding the role of GTP-binding regulatory proteins in the regulation of inositol phospholipid-specific phospholipase C effector enzymes in phagocytic leukocytes. Treatment of HL-60 granulocytes with pertussis toxin, under the conditions (500-2000 rig/ml; 3-4 h) routinely employed in our studies and those of other investigators (l-3,9-11,17), induced ADPribosylation of 85-90% of the major 40-41-kDa toxin substrate present in isolated HL-60 cell membranes (Fig. 3). Given the results of immunochemical measurements of Gprotein expression in HL-60 cells (6,7), this substrate presumably includes both the 40-kDa subunit of Gi., and the 41-kDa a-subunit of Gi+ FMLP-and P,-purinergic-induced activation of phospholipase C in such toxin-treated cells was inhibited by 91 and 78%, respectively (Fig. 1). The magnitudes of these inhibitory effects suggest strongly that a substantial fraction of the particular G-protein species (Gi+ Gi.:,, or both) that interact functionally with these receptors has been modified by the toxin treatment. However, as noted in our previous studies, there is a small but significant quantitative difference in the efficacies that characterize P2-purinergic-and FMLPstimulated inositol polyphosphate accumulation in these toxin-treated cells. This residual phospholipase C activation is sufficient to produce a rapid 2-fold increase in IP3 levels. In turn, this elevation in IP, produces near normal mobilization of Ca*+ stores by maximally active concentrations of UTP or ATP (data not shown).
Since 10-S% of the Gi.z/s pool in HL-60 cells treated with toxin for 3-4 h was not ADP-ribosylated (Fig. 3), it may be argued that additional blockade of P2-purinergic-induced sig- [3H]Inositol-labeled, differentiated HL-60 cells were incubated in the absence (control) or presence (PTX) of pertussis toxin for 3 h (500 rig/ml for Experiment 1; 400 rig/ml for Experiment 2). Membranes isolated from these cells were then incubated for 1 min (Experiment 2) or 2 min (Experiment 1) at 37 "C in the basic assay medium (described under "Experimental Procedures") containing either 350 nM free Ca2+ or 1 mM free Ca*+. Parallel samples also contained (in addition to 350 nM free Ca*+) no agonists (none); 1 &M GTP (GTP); 1 /LM GTP plus 10 PM FMLP (GTP/FMLP); 1 /LM GTP plus 1 mM Mg ATP (GTP/ATP); 1 FM GTP plus 1 mM MgATP plus 10 PM FMLP (GTP/ATP/FMLP); 1 PM GTP plus 30 PM UTP (GTP/ UTP); or 1 pM GTP plus 30 pM UTP plus 10 pM FMLP (GTP/UTP/ FMLP). Accumulation of [3H]inositol polyphosphates was quantified as described under "Experimental Procedures." Each data point represents an average of duplicate determinations. Percentage changes in total activity are defined as 1 -((agonist -none) naling requires modification of the residual pool of toxin substrate. In studying the ability of pertussis toxin to uncouple angiotensin II receptors from inhibition of adenylate cyclase in hepatocytes, Pobiner et al. (23) found that at least 90% of the membrane Gi pool had to be ADP-ribosylated to observe a 50% attenuation of the angiotensin action on cyclase activity in hepatocyte membranes.
Nearly 100% ADP-ribosylation was required to block completely the ability of the hormone to inhibit cyclic AMP accumulation in intact hepatocytes. Such results indicate an important role for receptor/ G-protein stoichiometry (particularly if [Gil >> [receptor]) in defining the functional consequences of pertussis intoxification. To achieve nearly 100% ADP-ribosylation of Gi.2,3 in HL-60 granulocytes required prolonged (>12 h) exposure to the toxin (Fig. 3). Despite the further reduction in unmodified Gi.213 content induced by this treatment, there was no additional decrease in the residual stimulation of inositol polyphosphate accumulation by either Pz-purinergic agonists or FMLP (Fig. 4). This observation suggests strongly that P,purinergic receptors might activate inositol phospholipid phospholipase C effector enzymes by mechanisms other than the primary pathway presumably mediated by unmodified Gi.213. In this regard, several studies have indicated that PZpurinergic receptors can activate inositol phospholipid phospholipase C effector enzymes via both pertussis toxin-sensitive and -insensitive mechanisms (24-30). This differential toxin sensitivity can be explained in part by cell-specific expression of P2-purinergic receptor subtypes with nucleotide selectivities different from those observed in HL-60 cells and neutrophils (24-28). However, receptors with a nucleotide selectivity identical to that observed in HL-60 cells have also been described in ovine and primate pituitary cells (29) and human fibroblasts (30). In the former cell types, activation of inositol polyphosphate accumulation was completely insensitive to pertussis toxin treatment.
In the toxin-treated fibroblasts, as in HL-60 cells, the effects of extracellular nucleotides were partially inhibited. The ability of a single Gprotein-coupled receptor subtype to utilize multiple G-proteins (pertussis toxin sensitive and insensitive) for the activation of phospholipase C effecters has also been suggested by Ashkenazi et al. (31) Tables I and II). Therefore, pertussis toxin treatment appears to attenuate some step in GTPdependent phospholipase C activation even in the absence of added receptor agonists. ADP-ribosylation of G&pe G-proteins in neutrophils is known to produce a well characterized inhibition of G-protein activation by occupied FMLP receptors (l-3, 9). Our results show that ADP-ribosylation of the relevant toxin-sensitive G-protein(s) also slows the GTPdependent cycle of G-protein activation in the absence of agonist-occupied receptors. Such inhibition has not been reported previously in studies of GTP-dependent phospholipase C activity in membranes derived from differentiated HL-60 cells or neutrophils (l-4). However, it is consistent with results from a recent study by McLeish et al. (32) showing that membranes isolated from pertussis toxin-treated HL-60 granulocytes exhibit a 65-70% reduction in the rate of high affinity GTPase and the rate of high affinity GTPyS binding measured in the absence of agonists. Similar findings have been reported in studies of neutrophil membranes (33-35). Such results suggest that ADP-ribosylation of a predominant GTP-binding protein in HL-60 or neutrophil membranes significantly slows the rate of GDP/GTP exchange even in the absence of agonist-occupied receptors. Attenuation of this exchange rate might reduce the steady-state level of activated G-protein produced in response to incubation with GTP per se and also to slow the rate but not extent of G-protein activation by the nonhydrolyzable GTP+. Consistent with this possibility, we observed that pertussis toxin inhibited only the initial rate (data not shown) but not the extent (Fig.  7) of GTPyS-induced phospholipase C activation in HL-60 cell membranes.
Thus, ADP-ribosylation appears to affect only the rate of G-protein activation but not the ability of the activated G-protein to stimulate phospholipase C. It should be stressed that these data and conclusions pertain only to the in situ function of the toxin-sensitive G-proteins in native HL-60 membranes. Such data and conclusions are not meant to imply that pertussis toxin treatment produces, or would produce, similar functional effects in purified preparations of the relevant G-proteins. Sunyer et al. (36) noted similar effects of ADP-ribosylation on agonist-independent but GTP-dependent inhibition of adenylate cyclase in S49 cyc-membranes. However, in additional studies utilizing purified Gi (70% Gi.3 and 30% Gi.2) reconstituted into lipid vesicles, these investigators observed no effects of ADP-ribosylation on GTPase activity or GDP release per se. They hypothesized that the toxin-induced inhibition of agonist-independent GTP-dependent signaling events observed in native cycmembranes may reflect G-protein activation by unoccupied receptors. Similar interaction of unoccupied receptors with Gi.2j3 in HL-60 membranes may explain the quite high rates of GTP-activated inositol polyphosphate release observed in the absence of added FMLP or Ps-purinergic agonist. The absolute rate and extent of inositol polyphosphate release induced by (UTP + GTP) in toxin-treated membranes was reduced by about 20-35% relative to that observed in control membranes  Tables I and II). However, we were surprised to observe that the ability of UTP (or ATP) to potentiate GTP-dependent phospholipase C was very similar in isolated membranes prepared from either control or pertussis toxin-treated cells. Thus, the dose-response curve characterizing UTP-induced activity in pertussis toxintreated membranes, while negatively shifted along the y axis, paralleled that observed in control membranes.
If it is assumed that P2-purinergic receptors activate phospholipase C exclusively through the mediation of a toxin-sensitive Gprotein(s) presumably Gi.2 or Gi-3), our results would suggest that ADP-ribosylation does not preclude interaction of this G-protein with occupied P2-purinergic receptors. This interpretation would suggest that Pz-purinergic receptors, but not FMLP receptors, contain structural features that facilitate some degree of interaction with the ADP-ribosylated cu-subunits of Gi-type proteins. Alternatively, as discussed previously, PP-purinergic receptors of the subtype expressed in HL-60 cells and neutrophils may have the capacity to activate phospholipase C effector enzymes via the mediation of a Gprotein(s) that is not a pertussis toxin substrate. It is also noteworthy that P2-purinergic-induced activation of inositol phospholipid breakdown in intact HL-60 cells was largely (80%) sensitive to inhibition by pertussis toxin treatment whereas in HL-60 membranes this same parameter was largely insensitive to toxin treatment. Conversely, in both intact cells (Fig. 1) and membranes   Table I), FMLP-stimulated phospholipase C activity was predominantly (70-90%) sensitive to toxin treatment.
It should also be noted that although both receptor agonists were equally efficacious in stimulating inositol polyphosphate accumulation in intact cells, FMLP was invariably less efficacious than UTP in the membrane assays   Table I). These observations suggest that some component of the FMLP receptor/G-protein/phospholipase C signaling cascade present in intact cells is lost or inactivated in the washed membrane preparation.
Several of the inositol phospholipid-specific phospholipase C enzymes appear to be either soluble proteins or only weakly associated with membranes (37). It is possible that the FMLP receptors in intact HL-60 cells activate, via Gi.z/s, a phospholipase C enzyme that is primarily cytosolic or very weakly associated with the plasma membrane. Given the substantial inhibition of UTP-induced ino-sit01 phosphate accumulation observed in intact cells, one can speculate additionally that P,-purinergic receptors, acting via Gi.2,3, also activate this phospholipase subtype. However, our results are consistent with the possibility that these receptors can also activate, via a pertussis toxin-insensitive mechanism, a distinct phospholipase C species that has relatively high avidity for the membrane. This putative Pz-purinergic-activated signaling pathway may predominate in studies of isolated membranes. McLeish et al. (32) have suggested recently that differences in the activation of Gi-2 (or additional G-proteins) by FMLP receptors and leukotriene B4 receptors may be responsible for differential activation of various neutrophil responses by these agonists. Similarly, FMLP but not ATP (or UTP) is a much more efficacious stimulus for superoxide production and primary granule secretion in neutrophils and HL-60 granulocytes (15)(16)(17). The differential activation of G-proteinregulated phospholipase signaling pathways may explain in part the disparate effects of these two species of Ca*-mobilizing agonists on the function of phagocytic leukocytes.

cells and HL-60 cell membranes. and chemotactic peptide-stimulated inositol phospholipid breakdown in HL-60
Pertussis toxin produces differential inhibitory effects on basal, P2-purinergic,