Elevated levels of cyclooxygenase-2 in antigen-stimulated mast cells is associated with minimal activation of p38 mitogen-activated protein kinase.

We have investigated possible factors that underlie changes in the production of eicosanoids after prolonged exposure of mast cells to Ag. Ag stimulation of cultured RBL-2H3 mast cells resulted in increased expression of cyclooxygenase (COX-2) protein and message. Other eicosanoid-related enzymes, namely COX-1, 5-lipoxygenase, and cytosolic phospholipase A(2) were not induced. Activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein (MAP) kinase preceded the induction of COX-2, whereas phosphatidylinositol 3' kinase and its substrate, Akt, were constitutively activated in RBL-2H3 cells. Studies with pharmacologic inhibitors indicated that of these kinases, only p38 MAP kinase regulated expression of COX-2. The induction of COX-2 was blocked by the p38 MAP kinase inhibitor SB202190, even when added 12-16 h after stimulation with Ag when p38 MAP kinase activity had returned to near basal, but still minimally elevated, levels. Interestingly, expression of COX-2 as well as cytosolic phospholipase A(2) and 5-lipoxygenase were markedly reduced by SB202190 in unstimulated cells. Collectively, the results imply that p38 MAP kinase regulates expression of eicosanoid-related enzymes, passively or actively, at very low levels of activity in RBL-2H3 cells. Also, comparison with published data suggest that different MAP kinases regulate induction of COX-2 in inflammatory cells of different and even similar phenotype and suggest caution in extrapolating results from one type of cell to another.

Induction of telomerase activity during development of human mast cells from peripheral blood CD34+ cells: comparisons with tumor mast-cell lines.

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+ /CD117+ /CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+ /CD117+ /CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.

Disruption of Raf-1/heat shock protein 90 complex and Raf signaling by dexamethasone in mast cells.

Antigen stimulation of mast cells via the IgE receptor, FcepsilonRI, results in the recruitment of the cytosolic tyrosine kinase, Syk, and the activation of various signaling cascades. One of these, the extracellular signal-regulated kinase (ERK2) cascade, is inhibited by low concentrations of the immunosuppressant drug, dexamethasone, probably at a step prior to the activation of Raf-1 (Rider, L. G., Hirasawa, N., Santini, F., and Beaven, M. A. (1996) J. Immunol. 157, 2374-2380). We now show that treatment of cultured RBL-2H3 mast cells with nanomolar concentrations of dexamethasone causes dissociation of the Raf-1.heat shock protein 90 (Hsp90) complex. Raf-1 bereft of this protein fails to associate with the membrane or Ras in antigen-stimulated cells. Upstream events such as the Syk-dependent phosphorylation of Shc, the engagement of Shc with the adapter protein, Grb2, and the activation of Ras itself are unaffected. Interestingly, the counterpart of Raf-1 in the c-Jun N-terminal kinase (JNK) cascade, MEKK-1 (mitogen-activated protein kinase/ERK kinase), is similarly associated with Hsp90, and this association as well as the activation of MEKK-1 are disrupted by dexamethasone treatment. Disruption of the ERK and JNK cascades at the level of Raf-1 and MEKK-1 could account for the inhibitory action of dexamethasone on the generation of inflammatory mediators in stimulated mast cells.

Compound 48/80 activates mast cell phospholipase D via heterotrimeric GTP-binding proteins.

Previous studies have indicated the presence of a cholera toxin-sensitive phospholipase D (PLD) in cultured RBL-2H3 mast cells that is synergistically activated via calcium, protein kinase C, and another unidentified signal. Here we identify a third potential signal for activation transduced by a pertussis toxin-sensitive trimeric GTP-binding protein, most likely via G(i2) or G(i3). Quercetin-treated RBL-2H3 cells in which expression of G(alphai2) and G(alphai3) is enhanced more than 7-fold respond to the G(i) stimulant compound 48/80 with the activation of PLD, a transient activation of phospholipase C, and enhanced membrane GTPase activity. The activation of PLD was blocked in pertussis toxin-treated cells and, as with other stimulants of PLD, was enhanced in cholera toxin-treated cells. The PLD response to compound 48/80 was only partially inhibited by calcium deprivation and inhibition of protein kinase C to indicate a component of the response that was independent of calcium, protein kinase C, and, presumably, phospholipase C. Based on these and other data, we hypothesized that betagamma-subunits, released from G(i2) or G(i3) by compound 48/80 or from G(s) by cholera toxin, provide an additional signal for the activation of PLD. Consistent with this hypothesis, recombinant G(beta2gamma2) subunits, but not G(alphai-3) subunits, at concentrations of 50 to 300 nM markedly synergized PLD activation by compound 48/80 in permeabilized RBL-2H3 cells.

Recognizing the pleckstrin homology domain fold in mammalian phospholipase D using hidden Markov models.

Phospholipase D was first described in plant tissue but has recently been shown to occur in mammalian cells where it is activated by cell surface receptors. Its mode of activation by receptors in unclear. Biochemical studies suggest that it may occur downstream of other effector proteins and that small GTP-dependent regulatory proteins may be involved. The sequence in a non-designated region of mammalian phospholipase D1 and 2 shows similarity to a structural domain that is present in signalling proteins that are regulated by protein kinases or heterotrimeric G-proteins. Mammalian phospholipase D has structural similarities with other lipid signalling phospholipases and thus may be regulated by receptors in an analogous fashion.

Inositol polyphosphates regulate the membrane interactions of the endosomal p100, G-protein-related protein.

The protein, p100, was previously identified as a G-protein related protein that cycles on and off the cytoplasmic face of the endosome membrane (Traub et al., Biochem. J. 280 (1991) 171-178). Here we present evidence that the inositol polyphosphates, inositol 1,4, 5-trisphosphate (IP3) and inositol hexakisphosphate (IP6), release p100 from light-density microsomal membranes and inhibit rebinding of p100 through receptors, which are specific for IP3 or for IP6. These receptors can be co-extracted with p100 from the microsomes by 0.5 M Tris-HCl and, in the soluble state, they exhibit similar binding activity towards the inositol polyphosphates as do untreated microsomes. Soluble p100 self-aggregates and this aggregation is blocked by both IP3 and IP6. Stimulation of permeabilized rat basophilic leukemia (RBL-2H3) cells with carbachol, via transfected muscarinic m1 receptors, results in increased levels of inositol polyphosphates and the quantitative release of p100 into the cytosol. This effect is reversible and cytosolic p100 rebinds to the membrane as the levels of inositol polyphosphates decline. These findings suggest that p100 may belong to a family of IP-binding proteins whose intracellular localization is determined by extracellular signals.

Thapsigargin-induced secretion is dependent on activation of a cholera toxin-sensitive and phosphatidylinositol-3-kinase-regulated phospholipase D in a mast cell line.

Release of secretory granules by rat RBL-2H3 mast cells is mediated primarily through activation of protein kinase C (PKC) and elevation of cytosolic free calcium ([Ca++]I). Here, we show that secretion was also dependent on the activation of a cholera toxin-sensitive phospholipase (PL) D in cells stimulated with thapsigargin. Wortmannin, LY294002, butanol, propranolol and Ro31-7549 inhibited responses to variety of secretagogues in a manner consistent with the notion that secretion was regulated by both PLD and PKC in a phosphatidylinositol-3-kinase-dependent manner. The effects of these inhibitors, however, were especially pronounced in cells activated by thapsigargin. This stimulant induced minimal stimulation of PLC but measurable activation of PLD, as assessed by formation of phosphatidylethanol in the presence of ethanol. The activation of PLD was suppressed by inhibitors of phosphatidylinositol-3-kinase and was dependent on a rise in [Ca++]i because thapsigargin failed to activate PLD and secretion when elevation of [Ca++]i was blocked. Treatment of cells with cholera toxin resulted in selective and similar enhancements in the activation of PLD and secretion by thapsigargin, whereas stimulation of PLC and PLA2 was unaffected. A role for PKC was indicated by the blockade of secretory response to thapsigargin by the PKC inhibitor Ro31-7549 and by the ability of the PKC agonist phorbol-12-myristate-13-acetate to reverse the inhibition of secretion by inhibitors of PLD. Such results suggested that thapsigargin, by causing substantial increases in [Ca++]I, induced secondary signals via PLD and PKC that synergized a calcium signal for secretion.

Quercetin sensitizes RBL-2H3 cells to polybasic mast cell secretagogues through increased expression of Gi GTP-binding proteins linked to a phospholipase C signaling pathway.

Polybasic secretagogues such as mastoparan, compound 48/80, substance P, and somatostatin stimulate secretion in rat peritoneal mast cells through direct activation of the heterotrimeric G protein, G(i-3). Cultured RBL-2H3 mast cells do not normally respond to these secretagogues, but, as reported here, they do so after prolonged exposure to the kinase inhibitor, quercetin. This inhibitor, which causes phenotypic changes in RBL-2H3 cells, induces a substantial increase (more than sevenfold) in the expression of alpha subunits of the pertussis toxin-sensitive G proteins, G(i-2) and G(i-3). Compound 48/80-induced secretion is associated with transient hydrolysis of phosphoinositides and a transient increase in cytosolic calcium ions. These responses are inhibited by pertussis toxin, and in addition, secretion is blocked by calcium chelation and the protein kinase C inhibitor, Ro31-7549. These results delineate a pathway for compound 48/80-induced secretion in mast cells via Gi protein(s), phospholipase C, calcium, and protein kinase C. The results also imply that phospholipase C, most likely phospholipase Cbeta3, can be transiently activated in RBL-2H3 cells by subunits of Gi proteins to induce cellular responses.

Mitogen-activated protein (MAP) kinase regulates production of tumor necrosis factor-alpha and release of arachidonic acid in mast cells. Indications of communication between p38 and p42 MAP kinases.

Aggregation of the high affinity IgE receptor (FcepsilonRI) in a mast cell line resulted in activation of the p42 and the stress-activated p38 mitogen-activated protein (MAP) kinases. Selective inhibition of these respective kinases with PD 098059 and SB 203580 indicated that p42 MAP kinase, but not p38 MAP kinase, contributed to the production of the cytokine, tumor necrosis factor-alpha, and the release of arachidonic acid in these cells. Neither kinase, however, was essential for FcepsilonRI-mediated degranulation or constitutive production of tumor growth factor-beta. Studies with SB 203580 and the p38 MAP kinase activator anisomycin also revealed that p38 MAP kinase negatively regulated activation of p42 MAP kinase and the responses mediated by this kinase.

Antigen activation of mitogen-activated protein kinase in mast cells through protein kinase C-dependent and independent pathways.

We demonstrate discrete pathways for activation of mitogen-activated protein (MAP) kinase in cultured RBL-2H3 mast cells through protein kinase C (PKC), cytosolic calcium, and a third pathway that provides sustained signals for activation in Ag-stimulated cells. Thus, p42 MAP kinase was activated by increasing intracellular free Ca2+ with thapsigargin or by stimulating PKC with PMA. The latter stimulation was selectively blocked by the protein kinase C inhibitor, Ro31-7549. Stimulation of p42 MAP kinase by Ag resulted in relatively sustained activation of MAP kinase which was only partially suppressed by Ro31-7549. Kinetic studies revealed two components of the MAP kinase response to Ag: a rapid but transient component that was Ro31-7549 sensitive and presumably PKC dependent; and a more sustained component that was Ro31-7549 resistant and presumably PKC independent. Similarly, Ro31-7549 inhibited the early but not late release of arachidonic acid, a finding that was consistent with the known regulation of phospholipase A2 by MAP kinase. Early tyrosine phosphorylation events which were thought to be essential for Ag-induced activation of p42 MAP kinase and release of arachidonic acid were unaffected by Ro31-7549. The findings suggested that release of arachidonic acid was regulated primarily through MAP kinase but that PKC may transiently influence this release, either directly or indirectly through MAP kinase.

Downstream signals initiated in mast cells by Fc epsilon RI and other receptors.

The significant contributions this past year to our understanding of IgE receptor (Fc epsilon RI) signaling in mast cells include studies with truncated Syk in a vaccinia expression system and Syk-negative variants of rat basophilic (RBL-2H3) cells. These studies demonstrate an essential role for Syk in initiating signals for secretion and release of arachidonic acid via phospholipase A2 and mitogen-activated protein kinase. A newly recognized addition to the repertoire of Fc epsilon RI-mediated signaling systems is the activation of sphingosine kinase, which contributes to calcium mobilization in mast cells. Advances have been made in our understanding of other receptors that regulate proliferation and differentiation of mast cells, and in our understanding of the ability of mast cells to mount acquired and acute responses to antigenic and bacterial challenge.

Constitutive and inducible mechanisms for synthesis and release of cytokines in immune cell lines.

We found that production and release of two functionally antagonistic cytokines, TGF-beta and TNF-alpha, were regulated differently in the mast cell, T cell, and macrophage cell lines RBL-2H3, MLA-144, and U-937, respectively. TGF-beta was produced and released constitutively in all three cell lines. When, however, the cell lines were stimulated with Ag, LPS, or calcium ionophore plus PMA, acceleration of release and some additional production of TGF-beta were apparent. In contrast, TNF-alpha was produced and released only when these lines were stimulated. Although neither the glucocorticoid, dexamethasone, nor the protein kinase C inhibitor, Ro31-7549, suppressed constitutive production or release of TGF-beta, these agents inhibited TNF-alpha production and the inducible component of TGF-beta production noted above. The release of these cytokines, whether constitutive or inducible, was dependent on Golgi-processing as indicated by inhibition with brefeldin A. Therefore, although both types of cytokines were processed by Golgi, only TNF-alpha and the inducible component of TGF-beta production were protein kinase C or steroid-regulated processes. These findings suggested that constitutive and inducible pathways exist for production and release of cytokines and that the inducible pathways can be selectively suppressed by pharmacologic agents.

Activation of the mitogen-activated protein kinase cascade is suppressed by low concentrations of dexamethasone in mast cells.

Antigen stimulation of mast cells via the IgE receptor, Fc epsilon RI, results in recruitment of the cytosolic tyrosine kinases, Lyn and Syk, and the phosphorylation of proteins. We examined the effects of the glucocorticoid dexamethasone on these events in a cultured (RBL-2H3) mast cell line. Nanomolar concentrations of dexamethasone suppressed phosphorylation of proteins that were associated with the activation of the mitogen-activated protein (MAP) kinase/phospholipase A2 pathway without inhibiting initial events. For example, tyrosine phosphorylation of the subunits of Fc epsilon RI, Lyn, or Syk or of the Ras-guanine nucleotide exchange factor, Vav, was not suppressed in cells treated with up to 1 microM dexamethasone. In contrast, phosphorylation of Raf1, MEK1, p42mapk, and cytosolic phospholipase A2, as well as the associated increase in MAP kinase activity and release of arachidonic acid, were markedly inhibited in cells treated with as little as 10 nM dexamethasone--a concentration that only partially inhibited hydrolysis of inositol phospholipids or release of secretory granules. Prolonged exposure to dexamethasone also resulted in a partial decrease in expression of MEK1, p42mapk, and cytosolic phospholipase A2, which may contribute further to the effects of dexamethasone on this pathway. Activation of the MAP kinase/phospholipase A2 pathway by the calcium-mobilizing agent thapsigargin was similarly suppressed in dexamethasone-treated cells. These findings suggested that an early step in the pathway, possibly a step immediately before the activation of Raf1, was suppressed by low concentrations of dexamethasone.

Enhancement of TNF-alpha synthesis by overexpression of G alpha z in a mast cell line.

Ag stimulation of mast cells via the IgE receptor (Fc epsilon RI) elicits production and release of numerous cytokines. This activation of Fc epsilon RI initiates various tyrosine kinase-dependent signaling cascades, which ultimately result in the de novo synthesis of cytokines. To date, no heterotrimeric G proteins have been implicated in this process. Here we report that the alpha subunit of the heterotrimeric G protein, Gz, can regulate production of the cytokine, TNF-alpha. The alpha subunit was overexpressed in a cultured mast cell line (RBL-2H3) known to contain G alpha z. In stimulated cells, overexpression of G alpha z significantly enhanced the production of TNF-alpha. This effect of G alpha z appeared to be restricted in that constitutive synthesis of the cytokine, TGF-beta, and Ag-stimulation of the phosphoinositide-dependent secretory pathway were not significantly affected. Thus, G alpha z, a heterotrimeric G protein, appeared to modulate the stimulatory pathways for induction of TNF-alpha synthesis in RBL-2H3 cells.

Calcium signalling: sphingosine kinase versus phospholipase C?

A recent study shows that sphingosine kinase and its lipid product have an essential signalling function; they act in the mobilization of calcium ions in antigen-stimulated mast cells. This finding may have relevance to signalling in other cells of the immune system.

Syk-dependent phosphorylation of Shc. A potential link between FcepsilonRI and the Ras/mitogen-activated protein kinase signaling pathway through SOS and Grb2.

Antigen receptors on T- and B-cells activate Ras through a signaling pathway that results in the tyrosine phosphorylation of Shc and the formation of a complex of Shc with the Grb2 adaptor protein. The high affinity receptor for immunoglobulin E (FcepsilonRI) in cultured mast (RBL-2H3) cells has been reported to function differently. Here we show to the contrary that engagement of FcepsilonRI with antigen leads to increased tyrosine phosphorylation of Shc and the association of Shc with Grb2 and other proteins (p120 and p140). Like the FcepsilonRI-mediated activation of the mitogen-activated protein kinase cascade, these responses are dependent on the tyrosine kinase Syk; they are enhanced by overexpression of Syk and are blocked by expression of dominant-negative Syk. Sos is constitutively associated with Grb2 in these cells but dissociates from Shc on stimulation with antigen. These reactions are rapid, reversible, and associated with the activation of Ras. Therefore, the Syk-dependent tyrosine phosphorylation of Shc and its association with Grb2 may provide a pathway through Sos for activation of Ras by FcepsilonRI.

Cloning of the cDNA encoding rat myosin heavy chain-A and evidence for the absence of myosin heavy chain-B in cultured rat mast (RBL-2H3) cells.

The complete amino acid sequence (1961 amino acids) of a vertebrate cellular myosin heavy chain-A was deduced from cDNA clones of a secretory rat mast cell line, the RBL-2H3 cell. The rat, human and chicken cellular myosin heavy chain-A exhibited high similarity in domains that allow binding of ATP and actin. The amino acid sequence of non-muscle myosin heavy chain-A from rat was 96% identical to that in human and 92% identical to that in chicken. Northern blot analysis of mRNA indicated the presence of single message of 7.4 kilobases. Northern blot, reverse-transcriptase polymerase chain reaction, and Western blot with isoform-specific antibodies indicated that RBL-2H3 cells expressed exclusively myosin heavy chain-A. Unlike rat PC12 cells, as well as a wide variety of other cultured cells and tissues, myosin heavy chain-B mRNA and protein were not detectable in RBL-2H3 cells. Because RBL-2H3 cells can be stimulated to release secretory granules as well as newly generated arachidonic acid and cytokines but lack myosin heavy chain-B, this cell line may provide a unique model to study the role of myosin heavy chain-A in cellular responses to antigen and other stimulants.

Sustained activation of phospholipase D via adenosine A3 receptors is associated with enhancement of antigen- and Ca(2+)-ionophore-induced secretion in a rat mast cell line.

The adenosine analog, N-ethylcarboxamidoadenosine (NECA), causes transient activation of phospholipase C and an enhancement of antigen-induced secretion in a rat mast cell (RBL-2H3) line via adenosine A3-receptors (Ramkumar et al., J. Biol. Chem. 268:16887, 1993) by a mechanism that is inhibited by bacterial toxins and potentiated by dexamethasone (Ali et al., J. Biol. Chem. 265:745-753, 1990). Here we show that NECA synergizes the secretory response to Ca(2+)-ionophore as well as to antigen. The ability of NECA to synergize the secretory responses persisted for 10 to 20 min, long after the early phospholipase C-mediated reactions to NECA had subsided. NECA caused, however, a dose-dependent sustained activation of phospholipase D, as indicated by the formation of [3H]phosphatidic acid, or in the presence of 0.3% ethanol, [3H]phosphatidylethanol. This activation was associated with a sustained increase in diglycerides, in protein kinase C activity and in the phosphorylation of myosin light chains by protein kinase C. The generation of diglycerides was enhanced in dexamethasone-treated cells and suppressed in cells that had been treated with cholera toxin or pertussis toxin. Collectively, the studies suggested that the generation of diglycerides via phospholipase D and the associated activation of protein kinase C were, by themselves, insufficient signals for secretion in RBL-2H3 cells, but that these reactions synergized responses to stimulants such as antigen or A23187 that caused substantial increases in [Ca2+]i.

A requirement for Syk in the activation of the microtubule-associated protein kinase/phospholipase A2 pathway by Fc epsilon R1 is not shared by a G protein-coupled receptor.

Stimulation of the mast cell line, RBL-2H3, with antigen via the tetrameric (alpha beta gamma 2) immunoglobulin E receptor (Fc epsilon R1) leads to the activation of cytosolic phospholipase A2 and the release of arachidonic acid. This pathway is dependent on the activation of the mitogen-activated protein (MAP) kinase. In this paper, we show that the MAP kinase/cytosolic phospholipase A2 pathway is linked to Fc epsilon R1 via the cytosolic tyrosine kinase, Syk, and that the GDP/GTP exchange factor, Vav, might be one candidate for accomplishing this link. Cross-linking of transmembrane chimeras containing the Fc epsilon R1 gamma motif, which is known to activate Syk, results in the tyrosine phosphorylation of Vav, activation of MAP kinase, and release of arachidonic acid. Cross-linking of chimeras containing the Fc epsilon R1 beta motif does not cause these events. Furthermore, stimulation of these events by antigen is enhanced by transient overexpression of a wild-type form of Syk and blocked by overexpression of a dominant negative form of Syk. By contrast, stimulation via the transfected, G protein-coupled, muscarinic m1 receptor is not influenced by either form of Syk and does not result in tyrosine phosphorylation of Vav. These data establish unequivocally that the two types of receptor are independently linked to the two types of receptor are independently linked to the MAP kinase/cytosolic phospholipase A2 pathway and demonstrate the existence of the Fc epsilon R1-Syk-MAP kinase pathway.

Activation of the mitogen-activated protein kinase/cytosolic phospholipase A2 pathway in a rat mast cell line. Indications of different pathways for release of arachidonic acid and secretory granules.

The role of mitogen-activated protein (MAP) kinase in the release of arachidonic acid was examined in a mutated mast cell (RBL-2H3(m1)) line that expressed both native Fc epsilon R1 and the G protein-coupled muscarinic m1 receptor. Stimulation of these cells with Ag, carbachol, Ca(2+)-ionophore, or thapsigargin resulted in the phosphorylation of Raf1, MEK1, p42mapk MAP kinase, and the recently cloned cytosolic phospholipase A2 (PLA2) and increased activities of both MAP kinase and PLA2, as well as release of arachidonic acid. Because this cascade of reactions was inhibited by guanosine 5'-(2-thiodiphosphate), it appeared to be dependent on a GTP-binding protein(s). These reactions, however, were not dependent on protein kinase C; the cascade was totally resistant to the actions of a selective protein kinase C inhibitor, Ro31-7549, whereas release of the secretory granule marker, hexosaminidase, was blocked by this agent. Differences between the stimulatory pathways for release of arachidonic acid and hexosaminidase were evident also from the effects of the kinase inhibitor, quercetin. The above cascade of reactions, including release of arachidonic acid, was inhibited by 50% with approximately 5 microM quercetin, whereas secretion was inhibited only at higher concentrations of inhibitor. Moreover, inhibition of the activation of MAP kinase and release of arachidonic acid were closely correlated. This and previous findings suggested that release of arachidonic acid was attributable to the regulation of cytosolic PLA2 by MAP kinase (for activation of PLA2) and Ca2+ (for association of PLA2 with the membrane), whereas release of hexosaminidase was regulated primarily by Ca2+ and protein kinase C.