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The Journal of Immunology, 2001, 167: 4805-4813.
Copyright © 2001 by The American Association of Immunologists

Activation of {beta}{gamma} Subunits of Gi2 and Gi3 Proteins by Basic Secretagogues Induces Exocytosis Through Phospholipase C{beta} and Arachidonate Release Through Phospholipase C{gamma} in Mast Cells

Xavier Ferry, Virginie Eichwald, Laurent Daeffler and Yves Landry1

Laboratoire de Neuroimmunopharmacologie, Institut National de la Santé et de la Recherche Médicale, Unité 425, Université Louis Pasteur-Strasbourg I, Faculté de Pharmacie, Illkirch, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mast cells are activated by Ag-induced clustering of IgE bound to Fc{epsilon}RI receptors or by basic secretagogues that stimulate pertussis toxin-sensitive heterotrimeric G proteins. The cell response includes the secretion of stored molecules, such as histamine, through exocytosis and of de novo synthesized mediators, such as arachidonate metabolites. The respective roles of G proteins {alpha} and {beta}{gamma} subunits as well as various types of phospholipase C (PLC) in the signaling pathways elicited by basic secretagogues remain unknown. We show that a specific Ab produced against the C-terminus of G{alpha}i3 and an anti-recombinant G{alpha}i2 Ab inhibited, with additive effects, both exocytosis and arachidonate release from permeabilized rat peritoneal mast cells elicited by the basic secretagogues mastoparan and spermine. A specific Ab directed against G{beta}{gamma} dimers prevented both secretions. Anti-PLC{beta} Abs selectively prevented exocytosis. The selective phosphatidylinositol 3-kinase inhibitor LY 294002 prevented arachidonate release without modifying exocytosis. G{beta}{gamma} coimmunoprecipitated with PLC{beta} and phosphatidylinositol 3-kinase. The anti-PLC{gamma}1 and anti-phospholipase A2 Abs selectively blocked arachidonate release. Protein tyrosine phosphorylation was inhibited by anti-G{beta}{gamma} Abs, LY294002, and anti PLC{gamma}1 Abs. These data show that the early step of basic secretagogue transduction is common to both signaling pathways, involving {beta}{gamma} subunits of Gi2 and Gi3 proteins. Activated G{beta}{gamma} interacts, on one hand, with PLC{beta} to elicit exocytosis and, on the other hand, with phosphatidylinositol 3-kinase to initiate the sequential activation of PLC{gamma}1, tyrosine kinases, and phospholipase A2, leading to arachidonate release.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mast cells play a central role in inflammatory and allergic reactions through the release of a variety of biologically active compounds, either stored in granules, i.e., histamine and proteases, or de novo synthesized following activation, i.e., arachidonic acid metabolites and multifunctional cytokines (1). The best-known physiological stimulus for mast cells is the cross-linking of the high affinity IgE receptor, Fc{epsilon}RI, by Ags involved in immediate-type allergic reactions and in the protection against parasitic infections (2). Connective tissue mast cells, called serosal mast cells, can also be triggered by a large number of polycationic molecules, collectively known as basic secretagogues (3). The latter include endogenous and exogenous amphiphilic peptides and drugs involved in various inflammatory processes (4). Cationic neuropeptides, such as substance P, constitute the major cause of neurogenic inflammation (5). Anaphylatoxin C3a, a fragment from the third component of complement, also belongs to the family of mast cell basic secretagogues (6), extending their interest in late immunological reactions. Other basic secretagogues include venom peptides such as mastoparan (4, 7, 8), polyamines such as spermine and compound 48/80 (9, 10), and many cationic drugs currently being used therapeutically, with adverse effects related to mast cell activation (11).

A crucial characteristic of the effect of basic secretagogues on mast cells is its sensitivity to pertussis toxin (8, 12, 13, 14), which is known to ADP-ribosylate a cysteine residue in the carboxyl terminus of {alpha} subunits from Gi, Go, and Gt proteins (15). Two pertussis toxin substrates have been proposed in rat peritoneal mast cells (16) and were identified as the Gi2 and Gi3 proteins (17, 18). The Gi3 protein has been proposed to be responsible for histamine secretion, since an Ab directed toward a decapeptide corresponding to the carboxyl terminus of its {alpha} subunit (G{alpha}i3) inhibited mast cell exocytosis (17). The involvement of Gi2 protein was unlikely, since an Ab directed against the decapeptide of the carboxyl terminus of G{alpha}t, considered an analog of G{alpha}i2 with 90% identity between them, failed to inhibit exocytosis (see Fig. 1Go). The respective roles of {alpha} and {beta}{gamma} subunits of pertussis toxin-sensitive G proteins in the signaling pathways of basic secretagogues have not been investigated. G{beta}{gamma} subunits introduced into permeabilized mast cells amplified secretion induced by Ca2+ and GTP{gamma}S, whereas G{alpha}i3 subunits had no effect (19).



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  		FIGURE 1. Sequence of carboxyl-terminus domain of G{alpha}i, G{alpha}t, and G{alpha}s proteins. Bold amino acids indicate the difference of homology between G{alpha}i2 and G{alpha}t proteins. Italic cysteine residues represent the sites ADP-ribosylated by pertussis toxin.

 
The mechanism by which basic secretagogues activate exocytosis and release of stored mediators appears to be distinct from the IgE-dependent pathway. Unlike IgE-induced exocytosis, secretion elicited by basic secretagogues is faster (seconds vs minutes). Antigenic cross-linking of IgE/Fc{epsilon}RI initiates the activation of Lyn and Syk tyrosine kinases followed by the Syk-mediated phosphorylation of phospholipase C{gamma}1 (PLC{gamma}1).2 PLC{gamma}1, in turn, catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol-1,4,5-trisphosphate (IP3) and diacylglycerol, mediating the release of intracellular calcium stores and protein kinase C (PKC) activation, which are both involved in exocytosis (2). Basic secretagogues initiate a rapid (5-s) production of IP3, indicating concomitant PLC activation and histamine secretion (14). The activation of PLC is not preceded by protein tyrosine phosphorylation (20). The subtype of PLC involved in this process remains unknown.

Following cytosolic phospholipase A2 (cPLA2) activation, arachidonate release reaches a maximum after 20- to 30-min incubation with IgE/Fc{epsilon}RI-dependent (21) or G protein-mediated triggers (22). The activation of cPLA2 was first considered to be a consequence of the increase in cytosolic calcium elicited by basic secretagogues and was proposed to be a prerequisite for histamine secretion (14, 23). However, alternative regulatory pathways can lead to cPLA2 activation, which requires calcium increase or phosphorylation by various protein kinases, including PKC and mitogen-activated protein kinase (MAPK) isozymes (24). Fc{epsilon}RI-induced cPLA2 activation is achieved by the p42 MAPK, independently of PKC (25, 26). Basic secretagogue-dependent activation of cPLA2 does not require p42/44 MAPK (20, 22), but is preceded by phosphatidylinositol 3-kinase (PI3K) and PKC-dependent activation of Syk kinase (27). The involvement of PLCs has not been studied in this pathway.

The present study was undertaken to assess the roles of both pertussis toxin-sensitive substrates Gi2 and Gi3 heterotrimeric G proteins, and their respective {alpha} and {beta}{gamma} subunits in the secretion of histamine (exocytosis) and arachidonate release induced by basic secretagogues in mast cells. Putative relationships between the pathways of stored and de novo-synthesized mediators were also considered through the involvement of phosphatidylinositol-3 kinase (PI3K), PLC{beta}, PLC{gamma}1, and cPLA2 to define the bifurcation point of these pathways. Here, we show that both Gi2 and Gi3 proteins are activated by basic secretagogues, allowing G{beta}{gamma} subunits to initiate exocytosis through the activation of PLC{beta}, on the one hand, and the release of arachidonate through the activation of PI3K leading to PLC{gamma}1 activation, on the other.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Mastoparan and spermine were purchased from Sigma (St. Louis, MO). Pertussis toxin was obtained from List Biological Laboratory (Campbell, CA). [3H]Arachidonic acid was purchased from Amersham Pharmacia Biotech (Little Chalfont, U.K.). Protein A-Sepharose and protein G-Sepharose beads were obtained from Amersham Pharmacia (Uppsala, Sweden). Protease inhibitor tablet cocktails were purchased from Roche Diagnostics (Mannheim, Germany). Anti-G{alpha}s (carboxyl-terminal 10 aa residues) and anti-recombinant G{alpha}i2 protein Abs were purchased from Chemicon International (Temecula, CA). Anti-G{alpha}i3 and anti-G{alpha}i2 (carboxyl-terminal 10 residues) Abs were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-G{alpha}t (carboxyl-terminal 10 residues) Ab and mAbs against p-Tyr (PY20) were purchased from Transduction Laboratory (Lexington, KY). Anti-G{beta} (carboxyl-terminal 20 aa of {beta}1 of mouse origin, with broad specificity to mouse, rat and human G{beta}1 to G{beta}4), anti-cPLA2 (amino-terminal domain), anti-PLC{beta} (carboxyl-terminal 10 aa), and anti-PLC{gamma}1 (epitope corresponding to aa residues 530–850 mapping within SH2-SH3 domains) Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Akt (Ser473) was purchased from Cell Signaling Technology (Beverly, MA).

Isolation and purification of mast cells

Male Wistar rats (Iffa-Credo, L’Arbesle, France), weighing 300–500 g, were stunned and bled. Twelve milliliters of balanced salt solution (HEPES buffer) containing 137 mM NaCl, 2.7 mM KCl, 0.3 mM CaCl2, 1.0 mM MgCl2, 0.4 mM NaH2PO4, 5.6 mM glucose, and 10 mM HEPES, NaOH to pH 7.4, and supplemented with 0.1% BSA were injected into the peritoneal cavity. The peritoneal fluid was collected and centrifuged for 3 min at 180 x g. The pellet was suspended in the same buffer, and mast cells were purified on a discontinuous BSA gradient (30 and 40%, w/v) as previously described (8). The pellet was resuspended in HEPES buffer, and cells were examined under a light microscope for purity (>97%). The trypan blue exclusion test indicated a viability >95%.

Permeabilization and determination of histamine release

Purified mast cells (3 x 104 cells/assay) were preincubated for 5 min at 37°C before permeabilization by adding streptolysin-O (0.4 U/ml). After 1 min Abs were added for 2 min. Then cells were triggered by basic secretagogues. Reactions were terminated 2 min later by addition of ice-cold buffer. The passive histamine release, in the absence of secretagogue, was <10% of the total content. The amount of histamine secretion was determined fluorometrically according to the method of Shore et al. (28) but without the extraction step.

Determination of arachidonate release

Purified mast cells were suspended in HEPES buffer (5 x 105 cells/ml) and incubated with 5 µCi/ml [3H]arachidonic acid for 2 h at 37°C. The cells were washed twice, resuspended in HEPES buffer (105 cells/assay), preincubated for 10 min, and triggered for 10 min at 37°C. The reaction was terminated by adding ice-cold buffer and placing the tubes on ice. Supernatants following centrifugation (180 x g, 3 min, 4°C) were collected and used to determine by liquid scintillation the amount of arachidonate released.

Determination of PI3K activation through Akt phosphorylation

Purified mast cells (5 x 105 cells/assay) were preincubated for 15 min at 37°C with vanadate (0.1 mM) in HEPES buffer and triggered with secretagogues. Reactions were terminated by adding ice-cold buffer and placing the tubes on ice. Cell pellets obtained after centrifugation (3 min, 180 x g, 4°C) were treated by adding lysis buffer (150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, protease inhibitor cocktail, and 20 mM Tris-HCl) and centrifuged (20 min, 12,000 x g, 4°C). Supernatants were suspended in 5x Laemmli buffer and boiled for 15 min. Then supernatants were resolved by 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes (Hybond ECL, Amersham). Membranes were saturated by incubation overnight in a blocking solution containing 100 mM NaCl and 0.1% casein (w/v), washed twice, and incubated for 1 h with primary anti-phospho-Akt Ab. After incubation with secondary Ab (anti-mouse IgG Ab conjugated to HRP) for 1 h, membranes were incubated for 2 min in ECL reagents (Amersham), and bound Abs were visualized by contact for 2 min with Kodak X-OMAT films (Eastman Kodak, Rochester, NY).

Determination of protein tyrosine phosphorylation

Supernatants of stimulated mast cell were prepared as described above and incubated for 24 h with 15 µl p-Tyr Ab (1 mg/ml); 40 µl protein G-Sepharose beads were added and incubated for 2 h at 4°C. Immunocomplexes were washed twice with lysis buffer, resuspended in 5x Laemmli buffer, boiled for 15 min, and centrifuged (2 min, 20,000 x g, 4°C). Supernatants were resolved by 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. Membranes were saturated by incubation overnight in a blocking solution containing 100 mM NaCl and 0.1% casein (w/v), washed twice, and incubated for 1 h with primary Ab raised against p-Tyr. After incubation with secondary Ab (anti-mouse IgG Ab conjugated to horseradish peroxidase) for 1 h, membranes were incubated for 2 min in ECL reagents, and bound Abs were visualized by contact for 2 min with Kodak X-OMAT films.

Immunoprecipitation of G{beta} and PLC{beta} or PI3K

Cell pellets were prepared and lysed as described above. Supernatants, mixed with 15 µl anti-PLC{beta}, anti-G{beta}{gamma}, or anti-PLC{gamma}1 Abs and with protein A-Sepharose beads were incubated overnight at 4°C. Immunocomplexes were washed, treated, and resolved by SDS-PAGE as described above. Nitrocellulose membranes were incubated for 1 h with primary Abs (anti-G{beta}, anti-PI3K, or anti-p-Tyr Abs). After incubation with secondary Ab (anti-rabbit or anti-mouse IgG conjugated to HRP) for 1 h, membranes were incubated for 2 min in ECL reagents, and bound Abs were visualized.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The in vitro activation of heterotrimeric G proteins by basic secretagogues involves their interaction with the carboxyl terminus of G protein {alpha} subunits (29, 30), explaining the interest in using selective Abs of corresponding peptide sequences. We chose the wasp venom peptide mastoparan (7, 8) as a member of cationic peptide and the natural polyamine spermine (9, 10) to represent other cationic secretagogues. Streptolysin-O creates pores through the cell membrane and allows entry of Abs into mast cells (31). Permeabilization was controlled in each experiment (results not shown) by monitoring secretion elicited by GTP{gamma}S, a nonhydrolysable analog of GTP that triggers heterotrimeric and small G proteins in permeabilized mast cells (32).

Involvement of Gi2 and Gi3 proteins in mast cells exocytosis and arachidonate release

We studied the effects of Abs directed against the C-terminal decapeptides (sequences shown in Fig. 1Go) from G{alpha}i3, G{alpha}t, and G{alpha}s on permeabilized mast cells. Anti-G{alpha}i3 Abs dose-dependently inhibited approximately 60% of histamine secretion elicited by mastoparan or spermine (Fig. 2GoA). Anti-G{alpha}t Abs did not modify the exocytotic response of mast cells to mastoparan or spermine (Fig. 2GoB). Similarly, Aridor et al. (17) showed that anti-G{alpha}i3 Abs inhibited exocytosis triggered by GTP{gamma}S, whereas anti-G{alpha}t Abs were inefficient. The Gs subtype of heterotrimeric G proteins may regulate mast cell exocytosis (33), but we observed no effect of anti-G{alpha}s Ab on the response to basic secretagogues (Fig. 2GoC).



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  		FIGURE 2. Effect of anti-G{alpha}i3 (A), G{alpha}t (B), or G{alpha}s (C) carboxyl-terminus Abs on histamine secretion induced by basic secretagogues. Cells were incubated with streptolysin-O (0.4 U/ml) for 1 min at 37°C before the addition of Ab. Mast cells were stimulated 2 min later by adding mastoparan (0.1 mM) or spermine (3 mM). One hundred percent of each stimulation stands for histamine secretion induced by each compound on permeabilized cells in the absence of Ab (A, mastoparan, 17.2 ± 0.6%; spermine, 32.5 ± 4.9%; B, mastoparan, 29.2 ± 1.7%; spermine, 21.5 ± 3.5%; C, mastoparan, 30.4 ± 6.4%; spermine, 21.1 ± 0.9% of total histamine content). Controls show the Ab effect on unstimulated permeabilized cells (A, 7.9 ± 1.8%; B, 7.9 ± 1.8%; C, 14.2 ± 8.0%). Values are the mean ± SEM of four independent experiments.

 
The above results confirmed the involvement of Gi3 in the triggering effects of basic secretagogues. To determine whether Gi2, the second pertussis toxin substrate characterized in rat peritoneal mast cells (17), was also involved, we used two different anti Gi2 Abs (Fig. 3Go, A and C). The anti-recombinant G{alpha}i2 protein Ab (Fig. 3GoA) prevented histamine secretion induced by basic secretagogues without modifying controls in the absence of mastoparan or spermine. These results allowed us to propose both Gi2 and Gi3 as targets of basic secretagogues. Accordingly, the simultaneous inactivation of Gi2 and Gi3 proteins by anti-G{alpha} Abs provided additive inhibition of histamine secretion elicited by basic secretagogues (Fig. 3GoB).



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  		FIGURE 3. Effects of anti-G{alpha}i2 recombinant protein and anti-G{alpha}i2 carboxyl-terminus Abs on histamine secretion induced by basic secretagogues. Experiments were performed as detailed in Fig. 2Go. A, One hundred percent of histamine secretion in the absence of Ab corresponded to 24.3 ± 5.8% (mastoparan) or 21.8 ± 1.8% (spermine) of the total histamine content; controls are the Ab effect on unstimulated permeabilized cells (7 ± 3%). B, Effects of anti-recombinant G{alpha}i2 and anti-G{alpha}i3 Abs (dilution, 1/1000) added alone or simultaneously. Histamine secretion induced by each compound in permeabilized cells without Ab was 40.8 ± 1.1% (mastoparan) or 28.3 ± 3.3% (spermine) of the total histamine content. C, One hundred percent histamine secretion in the absence of Ab corresponded to 24.3 ± 5.8% (mastoparan) or 21.8 ± 1.8% (spermine) of the total histamine content; controls are the Ab effect on unstimulated permeabilized cells calculated as a percentage of the total histamine content. D, Effect of pretreatment (2 h at 37°C) of mast cells with pertussis toxin (PTx; 50 ng/ml), and the effect of anti-G{beta} Ab (40 µg/ml; see Fig. 4Go) on histamine secretion induced by the Ab of the carboxyl terminus of G{alpha}i2 (shown as control in C). Values are the mean ± SEM of four independent experiments.

 
The anti-G{alpha}i2 C-terminus Ab (Fig. 3GoC) was apparently less efficient in preventing induced histamine secretion. However, this Ab led to histamine secretion in the absence of cationic secretagogues (control, Fig. 3GoC). This observation suggested that the anti-Gi2 C-terminus Ab might stimulate heterotrimeric G proteins, mimicking the basic secretagogue effect. To study this hypothesis we pretreated mast cells with pertussis toxin, which is known to ADP-ribosylate a cysteine residue in the G{alpha}i protein carboxyl terminus (see Fig. 1Go). Pertussis toxin pretreatment decreased the secretory response of mast cells (Fig. 3GoD). Thus, the anti-G{alpha}i2 C-terminus Ab mimics the effect of basic secretagogues characterized by pertussis toxin sensitivity. Also, histamine secretion elicited by this Ab (Fig. 3GoD) was prevented by anti-G{beta} Abs. Similarly, an anti-G{alpha}t C-terminus Ab has been reported to activate Gt protein (34).

The anti-recombinant G{alpha}i2 protein Ab and the anti-G{alpha}i3 C-terminus Ab both prevented arachidonate release induced by basic secretagogues (Fig. 4Go, A and B) with additive effects (inset in Fig. 4GoD). These data indicate that Gi2 and Gi3 are similarly involved in exocytosis and arachidonate release, indicating that Gi2 and Gi3 activation is a common step of the corresponding signaling pathways.



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  		FIGURE 4. Effect of anti-G{alpha}i2 recombinant protein and anti-G{alpha}i3 carboxyl-terminus protein on arachidonate release (A and B) and anti-G{beta} Abs on histamine secretion and arachidonate release (C and D) induced by basic secretagogues. A, One hundred percent induced arachidonate release from permeabilized cells in the absence of Ab was 1086 ± 52 dpm (mastoparan) or 1331 ± 63 dpm (spermine). B, One hundred percent induced arachidonate release from permeabilized cells in the absence of Ab was 1055 ± 26 dpm (mastoparan) or 1057 ± 20 dpm (spermine). C, One hundred percent induced histamine secretion in the absence of Ab was 39.7 ± 12.7% (mastoparan) or 38.6 ± 3.8% (spermine) of the total histamine content. D, One hundred percent of induced arachidonate release from permeabilized cells without Ab was 778 ± 21 dpm (mastoparan) or 813 ± 29 dpm (spermine). The inset in D shows the effects of pretreatment (2 h at 37°C) of mast cells with pertussis toxin (PTx; 50 ng/ml) and the effect of anti-G{beta} Ab (40 µg/ml) on arachidonate release. Controls stand for the Ab effect on unstimulated permeabilized cells (A, 486 ± 32 dpm; B, 487 ± 30 dpm; C, 7.0 ± 3.1%; D, 435 ± 14 dpm). Values are the mean ± SEM of four independent experiments.

 
Involvement of {beta}{gamma} subunits of G proteins in exocytosis and arachidonate release

Both {alpha} and {beta}{gamma} subunits of heterotrimeric G proteins can stimulate effectors (see Ref. 35 for review). To address the question of whether G{beta}{gamma} subunits were involved in signal transduction elicited by basic secretagogues, we studied the effect of an anti-G{beta} Ab with broad specificity to mouse, human, and rat G{beta}1 to G{beta}4 subunits. This Ab did not elicit mast cell secretory responses (controls, Fig. 4Go, C and D), but strongly inhibited histamine and arachidonate release (Fig. 4Go, C and D). These observations strongly suggest that {beta}{gamma} dimers of pertussis toxin-sensitive G proteins are involved in both exocytosis and arachidonic acid release induced by basic secretagogues in connective tissue mast cells.

Role of PLC{beta}, PLC{gamma}1, and cPLA2 in exocytosis and arachidonate release

The involvement of PLC in the secretory responses of mast cells to cationic triggers has been proposed by Nakamura and Ui (14), but the subtype of PLC has not been characterized. Considering the role of {beta}{gamma} dimers of heterotrimeric G proteins suggested by the above data, we assessed the involvement of PLC{beta} subtypes, with anti-PLC{beta}1, -{beta}2, and -{beta}3 Abs. We obtained similar results with all three Abs, calling into question their subtype selectivity. Although all PLC{beta} Abs were seen to prevent basic secretagogue-induced histamine secretion (Fig. 5GoA), none of them was able to modify induced arachidonic acid release (Fig. 5GoB). On the contrary, anti-PLC{gamma}1 Abs did not alter exocytosis (Fig. 5GoC), but prevented arachidonate release (Fig. 5GoD) triggered by mastoparan or spermine. The various PLC Abs studied had no effect on mast cells in the absence of cationic triggers (controls, Fig. 5Go).



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  		FIGURE 5. Effects of anti-PLC{beta}3, anti-PLC{gamma}1, and anti-cPLA2 Abs on exocytosis and arachidonate release induced by basic secretagogues. Cells were incubated with streptolysin-O (0.4 U/ml) for 1 min at 37°C, and Abs were added. Mast cells were stimulated by mastoparan (0.1 mM) or spermine (3 mM). Controls are the Ab effect on unstimulated permeabilized cells. One hundred percent values correspond to induced histamine secretion or induced arachidonate release from permeabilized cells in the absence of Ab: A, 6.8 ± 0.7% (control), 36.9 ± 4.2% (mastoparan), and 28.3 ± 4.9% (spermine) of total histamine content; B, 534.2 ± 23.4 dpm (control), 944.5 ± 28.5 dpm (mastoparan), and 1106.8 ± 34.1 dpm (spermine); C, 9.6 ± 0.5% (control), 31.0 ± 5.2% (mastoparan), and 33.2 ± 5.9% (spermine) of total histamine content; D, 476 ± 16 dpm (control), 1020 ± 33 dpm (mastoparan), and 1118 ± 16 dpm (spermine); E, 6.7 ± 0.7% (control), 25.9 ± 7.9% (mastoparan), and 29.5 ± 1.2% (spermine) of total histamine content; F, 667 ± 29 dpm (control), 1003 ± 32 dpm (mastoparan), and 1291 ± 26 dpm (spermine). Insets in A and B show the respective effects of anti-PLC{beta}1, -{beta}2, and -{beta}3 Abs (40 µg/ml) on secretions triggered by 0.1 mM mastoparan. Values are the mean ± SEM of four independent experiments.

 
The activity of cPLA2 generates arachidonate from membrane phospholipids. The activation of cPLA2 has been proposed to be a prerequisite for histamine secretion induced by basic compounds, suggesting its involvement in both secretion types (14, 23). However, we observed that anti-cPLA2 Abs selectively prevented arachidonate release without affecting exocytosis or cells in the absence of trigger (Fig. 5Go, E and F). These results show that the involvement of PLC{beta} is restricted to the exocytotic pathway, and that PLC{gamma}1 and cPLA2 are selectively involved in the arachidonate release pathway.

Sequence of events leading to exocytosis and arachidonate release

The above results suggested that the early step of basic secretagogue transduction is common to both signaling pathways, involving {beta}{gamma} subunits of Gi2 and Gi3 proteins. G{beta}{gamma} is known to be able to interact with PLC{beta} (35) and PI3K (36), increasing their activities. This suggests that PI3K might be one of the direct effectors of basic secretagogue-activated G proteins. Activation of PI3K results in local accumulation of PIP3 at the plasma membrane, allowing the recruitment of cytosolic proteins characterized by a pleckstrin domain (37). However, the involvement of PI3K in mast cell secretion is poorly documented.

The selective inhibitor of PI3K, LY294002 (38), inhibited arachidonate release, but did not modify histamine secretion (Fig. 6Go, A and B). The activation of PI3K by basic secretagogues is further demonstrated in Fig. 6GoC by the LY29402-sensitive phosphorylation of Akt (PKB), a common substrate for PI3K (39).



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  		FIGURE 6. Effects of LY294002, a PI3K inhibitor, on exocytosis, arachidonate release, and Akt (PKB) phosphorylation induced by basic secretagogues. A, Cells were preincubated for 15 min at 37°C with vanadate (0.1 mM) and the indicated concentrations of LY294002 and were stimulated for 10 min by spermine (3 mM) or mastoparan (0.1 mM). One hundred percent histamine secretion in the absence of LY294002 corresponds to 50.5 ± 2% (mastoparan) and 50.6 ± 3% (spermine) of the total histamine content. B, Cells were labeled with [3H]arachidonate as detailed in Materials and Methods. Then cells were preincubated for 15 min at 37°C with vanadate (0.1 mM) and the indicated concentrations of LY294002 and stimulated for 20 min by spermine (3 mM) or mastoparan (0.1 mM). One hundred percent arachidonate release in the absence of LY294002 treatment corresponds to 1153 ± 45 dpm (mastoparan) and 1098 ± 51 dpm (spermine). C, Phosphorylation of Akt (PKB) due to PI3K activation. Cells were preincubated for 15 min at 37°C with vanadate (0.1 mM) and the indicated concentrations of LY294002 and were stimulated for the indicated time with spermine (3 mM). Cell extracts were lysed by adding lysis buffer and were subjected to Western blotting with anti-phospho-Akt Abs. Data are representative of three separate experiments.

 
The interaction of G{beta}{gamma} with PLC{beta} and PI3K is confirmed by the experiments represented in Fig. 7Go. We observed by Western blotting the coimmunoprecipitation of G{beta} and PLC{beta} with the PLC{beta} serum (Fig. 7GoA), and the coimmunoprecipitation of PI3K and G{beta} with the anti-G{beta} serum (Fig. 7GoB). These observations allow us to propose G{beta}{gamma} as the bifurcation point of the two secretory pathways, with PLC{beta} as the first following member of the exocytotic pathway and PI3K as the first following member of the pathway leading to de novo-synthesized mediators.



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  		FIGURE 7. Western blotting of G{beta} from PLC{beta} immunoprecipitate (A), of PI3K from G{beta} immunoprecipitate (B), and of tyrosine-phosphorylated proteins from PLC{gamma}1 immunoprecipitate (C). Cells were preincubated for 15 min at 37°C with vanadate (0.1 mM) and stimulated by spermine (3 mM) for the indicated times. Cell extracts were immunoprecipitated, as indicated in Materials and Methods, by anti-PLC{beta} (A), anti-G{beta} (B), or anti-PLC{gamma}1 (C) Abs and subjected to Western blotting with anti-G{beta} (A), anti-PI3K (B), or anti-p-Tyr (C) Abs. Data are representative of three separate experiments.

 
The activation of PLC{gamma}1 can be achieved through its binding to membrane PIP3 generated by PI3K and/or through tyrosine phosphorylation (40, 41, 42). PLC{gamma}1 immunoprecipitate is shown in Fig. 7GoC. We did not observe any binding of anti-phosphotyrosine Abs to PLC{gamma}1, suggesting that tyrosine phosphorylation was not involved in its activation by spermine. This observation strongly suggested that protein tyrosine kinases (PTK) might be downstream of PLC.

The activation of mast cells by basic secretagogues in the presence of vanadate to inhibit protein tyrosine phosphatases leads to tyrosine phosphorylation of several cellular proteins with Mr of 26–100 kDa (20). These phosphoproteins included the p42/p44 MAPKs (22). Fig. 9Go shows that tyrosine phosphorylation of proteins of 36–57 kDa was prevented by anti-G{beta} Abs, LY294002, and anti-PLC{gamma}1 Abs. These data demonstrate that G proteins, PI3K, and PLC{gamma} are upstream of PTK.



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  		FIGURE 9. Proposed pathways for exocytosis and arachidonate release induced by basic secretagogues in serosal mast cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The sensitivity to pertussis toxin of the secretory responses of mast cells to the basic secretagogue compound 48/80 has been the first indication of the involvement of G proteins in this process (13, 14). This property has been extended to other basic triggers considered to stimulate mast cells in a receptor-independent manner by directly activating pertussis toxin-sensitive G proteins (4, 6, 7, 8, 9, 11, 12, 16, 17, 29, 30, 43, 44, 45). The direct activation of G proteins involves the ability of basic peptides and drugs to translocate across the plasma membrane into mast cells to reach their targets. This has been demonstrated for the neuropeptide substance P (46), but experimental evidence is lacking for other basic secretagogues. Thus, a role for some undetermined membrane receptor with low ligand selectivity, cannot be formally excluded. However, the receptor-mediated or receptor-independent stimulation of pertussis toxin-sensitive G proteins is an early step in the response of mast cells to basic secretagogues.

The present results allow us to propose both Gi2 and Gi3 as targets for basic secretagogues. Gi2 had been previously excluded (17), since anti-G{alpha}t Abs were unable to inhibit exocytosis, considering that anti-G{alpha}t Abs might recognize G{alpha}i2 due to the structural analogy of Gt and Gi2 C-terminals (Fig. 1Go). Gi3 was considered to mediate PLC-independent exocytosis, thus acting downstream of PLC, fulfilling the properties of the putative GE protein proposed by Gomperts (47, 48). It would be tempting to propose Gi2 and Gi3 as the GP and GE proteins acting in series to control stimulus-secretion coupling in mast cells (47). However, several observations argue against this being the case. Firstly, small G proteins, Rac2 and cdc42, have recently been considered as candidates for GE (49). Secondly, the cumulative inhibitory effect of anti-Gi2 and -Gi3 Abs (Fig. 3Go) is not compatible with the two proteins acting in series. Thus, we believe that both Gi2 and Gi3 correspond to the putative Gp protein, acting upstream of PLC and leading to exocytosis and arachidonate release.

Anti-G{beta} Abs fully inhibited exocytosis and arachidonate release (Fig. 4Go). This observation strongly suggests that {beta}{gamma} subunits of Gi2 and Gi3 play a major role in the transduction pathway. A regulatory role of {alpha} subunits cannot be excluded. Such a role would not involve adenylate cyclase inhibition, since pertussis toxin did not increase the cAMP level in mast cells (13).

The participation of PLA2 in exocytosis was first proposed by Nakamura and Ui (14). This was based on the inhibitory effect of mepacrine and p-bromophenacyl bromide, which are considered to be selective inhibitors of this enzyme, on both histamine secretion and arachidonate release triggered by compound 48/80. However, higher drug concentrations were required to inhibit histamine secretion than arachidonate release (14). Alternatively, Churcher et al. (50) proposed that PLA2 activation is not an essential precursor of histamine secretion, considering that under some circumstances exocytosis was observed without measurable release of arachidonate. The present results confirm the latter view; anti-cPLA2 Ab prevented arachidonate release without affecting histamine secretion (Fig. 5Go). Thus, the bifurcation point of the two pathways is localized upstream of cPLA2.

Basic secretagogues induce a rapid production of IP3, indicating concomitant PLC activation and histamine secretion (6, 8, 9, 14, 16). The present results confirm the participation of the PLC{beta} family in this process, as could be predicted from its ability to be activated by heterotrimeric G proteins (35). The coimmunoprecipitation of PLC{beta} and G{beta}{gamma} (Fig. 7GoA) confirms that PLC{beta} interacts with {beta}{gamma} subunits of Gi proteins. However, we were unable to distinguish between the different subtypes of PLC{beta} due to the lack of selectivity of the available Abs. More interestingly, the anti-PLC{beta} Abs were unable to prevent arachidonic acid release elicited by mastoparan or spermine. In contrast, PLC{gamma}1 appeared to be selective for the arachidonate release pathway initiated by basic triggers (Fig. 5Go). This constitutes a major difference from secretory processes elicited by Ags, where PLC{gamma}1 controls both exocytosis and arachidonate release (2). The involvement of PLC{beta} or PLC{gamma}1 in exocytosis triggered by basic secretagogues or Ags, respectively, might be responsible for the different kinetics of histamine secretion observed in each case. The direct coupling of PLC{beta} induces exocytosis within seconds, whereas the indirect coupling of PLC{gamma}1 to Fc{epsilon}R1 receptors leads to a delayed exocytosis. PLC{gamma}2 does not have a major role in the IgE/FceRI pathway (42). Experiments are in progress to determine its putative involvement in the basic secretagogue pathway.

At this point of our study PLC{gamma}1 can be placed upstream of cPLA2 and PKC in the activation order. The activation of PLC{gamma}1 can be achieved by tyrosine phosphorylation or by the interaction of its pleckstrin domain with membrane PIP3 generated by PI3K (35, 36). We did not detect any tyrosine phosphorylation of PLC{gamma}1 following mast cell stimulation by basic secretagogues (Fig. 7GoC), suggesting that the major stimulation of PLC{gamma}1 was achieved through its recruitment at the membrane to PIP3-rich domains. However, we cannot exclude a minor participation of tyrosine phosphorylation in the activation of PLC{gamma}. We propose that PLC{gamma}1 is localized downstream of PI3K, which generates PIP3. This is compatible with the recent observation that PI3K can be activated through its interaction with {beta}{gamma} subunits of trimeric G proteins (51, 52). The coimmunoprecipitation of PI3K and G{beta}{gamma} (Fig. 7GoB) prompts us to propose a direct interaction between G{beta}{gamma} and PI3K (Fig. 9Go).

The participation of PTKs has recently been proposed in the arachidonate pathway (20, 53), including Syk kinase (27). The observation that anti-PLC{gamma}1 Abs decrease tyrosine phosphorylation (Fig. 8Go) indicates that PLC{gamma}1 precedes PTK.



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  		FIGURE 8. Inhibition of tyrosine protein phosphorylation by anti-G{beta} Ab (A), LY294002 (B), or anti-PLC{gamma}1 Ab (C) elicited by mastoparan (0.1 mM) or spermine (3 mM) for 20 min in rat peritoneal mast cells. A, Cells were preincubated with vanadate (0.1 mM) for 15 min at 37°C and permeabilized for 1 min before adding G{beta} Ab, and cells were stimulated. Cell extracts were lysed 2 min later, and phosphotyrosine proteins were resolved by Western blotting. B, Cells were preincubated with vanadate (0.1 mM) and LY294002 for 15 min prior to stimulation. Cell extracts were immunoprecipitated with anti-p-Tyr Ab, and phosphotyrosine proteins were resolved by Western blotting. C, Cells were preincubated with vanadate (0.1 mM) for 15 min at 37°C and permeabilized for 1 min before addition of anti-PLC{gamma}1, and cells were stimulated 2 min later. Cell extracts were immunoprecipitated with anti-p-Tyr Ab, and phosphotyrosine proteins were resolved by Western blotting. Data are representative of three separate experiments.

 
In summary, we propose a sequence of signaling events (Fig. 9Go) initiated by basic secretagogues in mast cells with the respective roles of PLC{beta} and PLC{gamma}1 in the control of exocytosis and arachidonate release. These two pathways elicited by a common stimulus suggest a selective subcellular localization for each PLC compatible with their respective roles. PLCs are known to generate IP3 and diacylglycerol, leading to the increase in cytosolic Ca2+ and to PKC activation. As we show that PLC{gamma}1 precedes PTK, the activation of PTK might require Ca2+ increase and/or PKC activation. These points need to be thoroughly studied.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Yves Landry, Faculté de Pharmacie, bp 24, 67401 Illkirch Cedex, France. E-mail address: landry{at}pharma.u-strasbg.fr Back

2 Abbreviations used in this paper: PLC, phospholipase C; cPLA2, cytosolic phospholipase A2; IP3, inositol-1,4,5-trisphosphate; G protein, heterotrimeric GTP-binding protein; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-triphosphate; PKB, protein kinase B (Akt); PKC, protein kinase C; PTK, protein tyrosine kinase. Back

Received for publication April 11, 2001. Accepted for publication August 1, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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