Differential activation of membrane phospholipid turnover by compound 48/80 and ionophore A23187 in rat mast cells

Membrane phospholipid turnover was investigated during histamine release from rat mast cells. Addition of calcium ionophore A23187 (0.5 microgram/ml) to mast cells prelabeled with [3H]glycerol induced the rapid and progressive increase in phosphatidic acid (PA) and 1,2-diacylglycerol (DG), which was concomitant with the small rise in phosphatidylinositol (PI). Loss of the level in triacylglycerol (TG) was very marked. Polyamine compound 48/80 (5 micrograms/ml) was shown to cause rises in PA, 1,2-DG, and PI without any significant changes in TG. Both stimuli increased incorporation of exogenous [3H]glycerol into phospholipids, indicating the involvement of de novo synthesis in phospholipid metabolism. Studies with [3H]arachidonic acid-labeled mast cells showed an enhanced liberation of radioactive arachidonate and metabolites upon histamine release. There were associated decreases of radioactivity in phosphatidylcholine (PC) and TG when exposed to A23187, while phosphatidylethanolamine (PE) was degraded as a result of 48/80 activation. The transient increases of [3H]arachidonoyl-1,2-DG and PA were caused by 48/80, while A23187 showed a gradual rise in the radioactivity in these two lipid fractions. These findings reflect activation of phospholipase C. When mast cells were activated by low concentrations of A23187 (0.1 microgram/ml) and 48/80 (0.5 microgram/ml), different behaviors of PI metabolism were observed. An early degradation of PI and a subsequent formation of 1,2-DG and PA suggest that the lower concentrations of these agents stimulate the PI cycle initiated by PI breakdown rather than de novo synthesis. These results demonstrate that marked and selective changes in membrane phospholipid metabolism occur during histamine release from mast cells, and that these reactions seem to be controlled by the coordination of degradation and biosynthesis, depending on the type and the concentration of stimulants. A23187 stimulates arachidonate release perhaps via the cleavages of PC and TG, whereas 48/80 liberates arachidonate from PE.     

Role of Ca2+ in phosphatidylinositol response and arachidonic acid release in formylated tripeptide- or Ca2+ ionophore A23187-stimulated guinea pig neutrophils

The role of Ca2+ in phospholipid metabolism and arachidonic acid release was studied in guinea pig neutrophils. The chemotactic peptide formylmethionyl-leucyl-phenyl-alanine (fMLP) activated [32P]Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) without any effects on the labeling of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). This activation was observed in Ca2+-free medium. Even in the neutrophils severely deprived of Ca2+ with EGTA and Ca2+ ionophore A23187, the stimulated labeling was not inhibited. When [3H]arachidonic acid-labeled neutrophils were stimulated by fMLP, a loss of [3H]arachidonic acid moiety in PI and the resultant increase in [3H]arachidonyl-diacylglycerol (DG), -PA, and free [3H]arachidonic acid was marked within 3 min. With further incubation, a loss of [3H]arachidonic acid in PC and PE became significant. These results suggest the activation of phospholipase C preceded the activation of phospholipase A2. In Ca2+-free medium, the decrease in [3H]arachidonyl-PI and the increase in [3H]arachidonyl-PA were only partially inhibited, although the release of [3H]arachidonic acid and a loss of [3H]arachidonyl-PC and -PE was completely blocked. These results show that PI-specific phospholipase C was not as sensitive to Ca2+ deprivation as arachidonic acid cleaving enzymes, phospholipase A2, and diacylglycerol lipase. Ca2+ ionophore A23187, which is known as an inducer of secretion, also stimulated [32P]Pi incorporation into PI and PA, although the incorporation into other phospholipids, such as PC and PE, was inhibited. This stimulated incorporation seemed to be caused by the activation of de novo synthesis of these lipids, because the incorporation of [3H]glycerol into PA and PI was also markedly stimulated by Ca2+ ionophore. But the chemotactic peptide did not increase the incorporation of [3H]glycerol into any glycerolipids including PI and PA. Thus, it is clear that fMLP mainly activates the pathway, PI leads to DG leads to PA, whereas Ca2+ ionophore activates the de novo synthesis of acidic phospholipids. When [3H]arachidonic acid-labeled neutrophils were treated with Ca2+ ionophore, the enhanced release of arachidonic acid and the accumulation of [3H]arachidonyl-DG, -PA with a concomitant decrease in [3H]arachidonyl-PC, -PE, and -PI were observed. Furthermore, the Ca2+ ionophore stimulated the formation of lysophospholipids, such as LPC, LPE, LPI, and LPA nonspecifically. These data suggest that Ca2+ ionophore releases arachidonic acid, unlike fMLP, directly from PC, PE, and PI, mainly by phospholipase A2. When neutrophils were stimulated by fMLP, the formation of LPC and LPE was observed by incubation for more than 3 min. Because a loss of arachidonic acid from PI occurred rapidly in response to fMLP, it seems likely the activation of PI-specific phospholipase C occurred first and was followed by the activation of phospholipase A2 when neutrophils are activated by fMLP...     

Concerted stimulation of PI-turnover, Ca2+-influx and histamine release in antigen-activated rat mast cells

Phospholipid metabolism in rat mast cells activated by antigen was examined with reference to phosphatidylinositol (PI) turnover. Upon antigen stimulation, histamine release from passively sensitized mast cells with IgE was potentiated by adding phosphatidylserine (PS). The addition of antigen to [3H]glycerol-prelabeled and sensitized mast cells induced a marked loss of radioactivity of PI and a concurrent accumulation of 1,2-diacylglycerol (DG) and phosphatidic acid (PA) within 5 to 60 sec. Furthermore, this antigen-induced PI breakdown was enhanced in the presence of Mg2+. Histamine release occurred in parallel with PI breakdown. On the other hand, the transient Ca2+ influx into mast cells, as measured by uptake of 45Ca2+, was found to occur quickly after cells were activated by antigen, which was concerted with PI breakdown. These results suggest that enhanced PI turnover may be an important step in the biochemical sequence of events leading to release of histamine, and that not only Ca2+ but also Mg2+ appears to take a part in stimulus-response coupling in rat mast cells.     

Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Secretogogue-stimulated phosphatidylinositol breakdown in the exocrine pancreas liberates arachidonic acid, stearic acid, and glycerol by sequential actions of phospholipase C and diglyceride lipase

When mouse pancreatic "minilobules" prelabeled with either [14C]arachidonic acid (AA), [14C]stearic acid (SA), or [3H]glycerol were stimulated with the secretogogue, caerulein, there was a 60-70% loss in radioactivity in phosphatidylinositol (PI) at 30 min. This loss was accompanied by the formation of [14C] phosphatidic acid (PA), [14C]diacylglycerol (DG), [14C] triacylglycerol (TG), and free [14C]AA, [14C]SA, and [3H]glycerol. The loss in radioactive PI was the same as the loss in chemically measured PI-phosphorus. Thirty to fifty per cent of the caerulein-induced loss of prelabeled PI could be accounted for as free [14C]AA, [14C]SA, or [3H]glycerol. Increased incorporation of fatty acid or glycerol residues into DG, PA, and TG accounted for the balance of the loss in PI. The specific DG-lipase inhibitor, RHC 80267, markedly inhibited the caerulein-stimulated release of [14C]AA, [14C]SA, and [3H]glycerol and roughly doubled the caerulein-induced increment in [14C]AA-, [14C]SA-, or [3H]glycerol-labeled DG, showing that the source of the caerulein-induced increment in fatty acids and glycerol was DG. When the PI was prelabeled with either [32P] orthophosphate, [3H]myoinositol, or [3H]glycerol, only 1% or less of the radioactivity in PI was in lysophosphatidylinositol (LPI), and there was no increase in radioactivity in LPI on stimulation with caerulein. These observations, taken together, argue strongly for a phospholipase C-catalyzed breakdown of PI followed by DG-lipase and argue against any significant involvement of phospholipase A2 in PI degradation in mouse pancreas. The formation of substantial amounts of free [14C]AA on stimulation supports the view that, among other things, the phosphoinositide effect in the exocrine pancreas serves to generate arachidonate (and its metabolites). The release of appreciable amounts of free fatty acids and glycerol shows that a significant portion of the DG formed as a result of caerulein-stimulated PI breakdown is not conserved in the phosphoinositide cycle.     

Strain differences in histamine release from mouse peritoneal mast cells induced by compound 48/80 or A23187

Strain differences among BALB/c, BDF1, CDF1, C3 H/He, C57 BL/6, DBA/2, ddy and ICR mice were investigated with respect to the ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80, a Ca2+ dependent histamine releaser, and the Ca2+ ionophore A23187. The ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80 were found to be high in BALB/c, ddY and ICR mice, but low in BDF1, CDF1, C3 H/He, C57 BL/6 and DBA/2 mice. Those induced by Ca2+ ionophore A23187 were high in BALB/c, BDF1, CDF1, C3 H/He, DBA2, ddy and ICR mice but low in C57 BL/6 mice. These results indicate that differences in histamine release from mouse peritoneal mast cells are strain dependent.     

An enhanced incorporation of fatty acid into phosphatidyl choline that parallels histamine discharge in mast cells

Summary Purified rat peritoneal and pleural mast cells preincubated briefly with radioactively labeled fatty acid were treated with A23187, which bypasses primary receptors in stimulating exocytosis. An enhanced incorporation of fatty acid into phosphatidyl choline (PC) that occurred in parallel with histamine release at 24–25°C was observed and was initially proportional to the total amount of histamine discharged. Enhanced PC labeling and histamine secretion were also proportional at temperatures ranging from 17–37°C. Both radioactive linoleic and palmitic acids were incorporated selectively at the -position of the glycerol backbone of PC. PC labeling by [³H]choline was not detectably different in control and stimulated cells, and phosphatidic acid did not exhibit selectively enhanced -acylation. Thus, the stimulated labeling in A23187-treated cells may occur secondary to the action of a phospholipase A₂ that favors PC as a substrate.Other peritoneal cell types exhibit a very similar A23187-stimulated selective labeling of PC. Therefore, autoradiography has been used to provide a direct demonstration that in purified preparations, mast cells are the principal cell type engaged in A23187-elicited incorporation of fatty acid into PC. The efficacy of this approach has relied on special procedures devised to obtain significantly different autoradiographic grain densities between control and stimulated preparations that can be attributed to differences in the level of [³H]palmitate-labeled PC.Preliminary tests using compound 48/80 as a secretory stimulus for mast cells have identified a similar selectively enhanced PC labeling. In either case, however, consideration of possible relationships between PC metabolism and the secretory process are premature since they have not been tested directly.     

Antigen-induced biphasic diacylglycerol formation in RBL-2H3 cells: the late sustained phase due to phosphatidylcholine hydrolysis is dependent on protein kinase C

Exposure to antigen (Ag) caused a biphasic 1,2-diacylglycerol (DG) production in [3H]myristic acid-labeled RBL-2H3 cells; the early, small transient phase and the second large sustained phase. The accumulation of phosphatidic acid (PA) or phosphatidylethanol (PEt) in the presence of ethanol was paralleled by the second-phase DG generation. Ag-induced formation of phosphocholine and choline in [3H]choline-labeled cells suggested the hydrolysis of phosphatidylcholine (PC) by phospholipases C and D. Treatment with phorbol myristate (PMA) or A23187 caused increases in [3H]DG and water-soluble [3H]choline metabolites. In protein kinase C (PKC) down-regulated cells, PEt formation was markedly reduced. In these cells DG production induced by Ag and A23187 was largely suppressed, thus indicating that PKC would play an important regulatory role for PC hydrolysis. However, because the A23187 treatment showed significant accumulation of water-soluble choline metabolites in PKC down-regulated cells, an increase in intracellular Ca2+ is another factor regulating PC hydrolysis. Taken together, these results may indicate that PC hydrolysis in response to Ag is dependent on PKC and Ca2+.     

Synergistic enhancement of histamine release from rat peritoneal mast cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate is not reflected by corresponding changes in phospholipid turnover

In an attempt to elucidate further the relationship between changes in phospholipid metabolism in, and histamine secretion from, purified rat peritoneal mast cells, the effects of the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) on these responses in stimulated and unstimulated cells was investigated. TPA caused a dose-dependent increase in the incorporation of 32PO4(3-) into the mast cell phospholipids; phosphatidic acid (PA) and phosphatidylcholine (PC), but not phosphatidylinositol (PI). TPA synergistically enhanced histamine release from cells stimulated by anti-immunoglobulin E (IgE) and the calcium ionophore A23187, reducing its ED50 from 150 nM to 40 nM, but did not alter histamine release from cells stimulated by compound 48/80. The effect of TPA on the changes in 32PO4(3-) incorporation into phospholipids associated with the above secretagogues did not, however, correlate well with the observed effects on histamine secretion induced by the same secretagogues. These observations are discussed in relation to the known effects of phorbol esters upon both secretory processes and phospholipid metabolism in other tissues.     

Inhibitory effect of diethylstilbestrol on histamine release by rat mast cells and its relation to the cellular ATP content

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.     

Histamine release by pharmacological agents in the absence of external free Ca2+

The involvement of extracellular free Ca2+ in histamine release was investigated in rat peritoneal mast cells. Incubation of non-antigenized cells in a media with high extracellular potassium did not increase histamine release. Secretion induced by A23187 and compound 48/80 in the presence of Ca2+ requires metabolic energy. In the absence of external free Ca2+ (2.5 microM) histamine release induced by A23187 is reduced but not abolished. Secretion induced by compound 48/80 is independent of extracellular Ca2+. These results lead us to suggest that mast cell plasma membranes probably lack voltage-gated Ca2+ channels and that external Ca2+ may not be an absolute requisite for histamine secretion.     

Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation

This study evaluated the effect of inhibitors of transmethylation on histamine release from rat mast cells and rat basophilic leukemia cells. IgE-mediated histamine release from rat basophilic leukemia cells (RBL-2H3 cells) was inhibited by 3-deazaadenosine (DZA) in the presence of L-homocysteine thiolactone (Hcy) or the combination of adenosine, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), and Hcy in a dose-dependent fashion. There were no significant changes in the cellular cAMP levels by these inhibitors. Histamine release induced by anti-IgE or dextran from normal rat mast cells was also blocked by DZA plus Hcy in a dose-dependent manner. DZA at 10(-3) M in the presence of 10(-4) M Hcy or the combination of 10(-3) M adenosine, 10(-4) M EHNA, and 10(-3) M Hcy inhibited lipid (perhaps phospholipid) methylation into RBL-2H3 cells without affecting choline incorporation. In the presence of 10(-3) M DZA plus 10(-4) M Hcy there was a 170-fold increase in [35S]AdoHcy with the concomitant appearance of 3-deaza-AdoHcy when the cells were incubated with [35S]methionine, thus indicating that these drugs inhibited methylation reaction(s) through the intracellular accumulation of AdoHcy and 3-deaza-AdoHcy. In contrast, histamine release from rat mast cells induced by the calcium ionophore A23187, compound 48/80, polymyxin B, or ATP was not inhibited by these compounds. These results suggest that IgE- or dextran-mediated histamine release involves methylation reactions(s), whereas the other secretagogues bypass this early step.     

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes.

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glucose inhibits insulin release when not promoting the entry of calcium into the beta-cells

The effect of antigen on the metabolism of polyphosphoinositides was investigated in sensitized rat peritoneal mast cells. Addition of antigen to rat peritoneal mast cells prelabelled with [3H]arachidonic acid resulted in a very rapid decrease in the level of phosphatidylinositol 4-phosphate (DPI) within 5 sec, which appeared to precede the breakdown of phosphatidylinositol (PI), while there was no significant decline of PI 4,5-bisphosphate (TPI). The reduced levels of these phosphoinositides returned almost to control or even slightly higher values by 300 sec in parallel with the antigen-stimulated [32P]phosphate incorporation into these lipids. This early and transient disappearance in DPI prior to that in PI was also observed in [3H]glycerol-prelabelled cells. These data suggest that DPI degradation upon stimulation by antigen in mast cells may be an initial step in the histamine release process.     

Evidence of a role for phosphatidylinositol synthesis in human amnion cell proliferation.

Phosphatidylinositol (PtdIns) is the key precursor of phosphoinositide-derived intracellular mediators. The effects of changing the rate of PtdIns synthesis on mitogenic activity of human amnion-derived WISH cells were investigated. Incubation of the cells with [3H]inositol caused a time- and dose-dependent PtdIns labeling. Exogenous Ca2+ inhibited [3H]inositol incorporation in a dose-dependent fashion; half-maximal inhibition occurred with 0.3-1.0 mM Ca2+. In contrast, removal of cytosolic Ca2+ by ionophore A23187 and 1 mM EGTA induced enhancement of the PtdIns labeling as a function of A23187 concentration, perhaps through release of inhibitory effects of endogenous Ca2+. The A23187-stimulated PtdIns labeling with [3H]inositol was not abolished by additional unlabeled inositol, suggesting that [3H]inositol labeling of PtdIns occurred mainly through de novo synthesis catalyzed by PtdIns synthase (EC 2.7.8.11). In cells with PtdIns synthase activity decreased by exogenous Ca2+, [3H]thymidine incorporation was also inhibited, while A23187 caused dose-dependent enhancement of thymidine incorporation. The changes in PtdIns synthase activity occurred in parallel with changes in mitogenic activity caused by increasing the dose of exogenous Ca2+ or A23187. A similar lowering of mitogenic activity was observed upon suppression of PtdIns synthase by pemirolast potassium (9-methyl-3-1H-tetrazol-5yl-4H-pyrido[1,2-a]pyridin-4-one potassium) via a Ca(2+)-independent mechanism. These data demonstrate that changes in PtdIns synthase activity by some agents acting via different mechanisms are associated with parallel changes in thymidine incorporation, and suggest that PtdIns production is tightly coupled to cell proliferation in human amnion cells.     

Altered signal transduction in erbB-transformed cells. Implication of enhanced inositol phospholipid metabolism in erbB-induced transformation

To clarify the signal transduction mechanism of the erbB gene (virus oncogene) products leading to cell growth and transformation, the alteration of signal transduction induced by enhanced inositol phospholipid metabolism was studied in chick embryo fibroblast cells (CEF cells) transformed by gag-fused erbB gene-carrying virus (GEV cells). The incorporations of 32P into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate were markedly increased in GEV cells. In GEV cells, the activities of lipid kinases such as phosphatidylinositol (PI), PIP, and diacylglycerol (DG) kinases were also increased. The activities of other important enzymes involved in inositol phospholipid metabolism, such as CDP-DG:myo-inositol transferase and phospholipase C, were not changed in GEV cells. Increased inositol phospholipid metabolism might lead to the production of second messengers, such as 1,2-DG and inositol 1,4,5-trisphosphate. Indeed, the 1,2-DG content was also increased in GEV cells. Moreover, the activity of protein kinase C (the Ca2+/phospholipid-dependent enzyme), which should be stimulated by 1,2-DG, was elevated in GEV cells; the protein kinase C activity in the membrane fraction of GEV cells was especially high. When CEF cells were treated with tetradecanoylphorbol acetate, protein kinase C activator, plus Ca2+ ionophore, [3H]thymidine incorporation was markedly stimulated, and maximal stimulation was observed with 1 nM Ca2+ ionophore A23187 plus 100 nM TPA. On the other hand, when GEV cells were treated with TPA plus Ca2+ ionophore A23187, [3H]thymidine incorporation was consistently inhibited. Next, studies were made to determine whether the erbB gene product itself had kinase activity on PI, PIP, and DG after membranes were mildly solubilized with Triton X-100 to prevent inactivation of these kinases. Immunoprecipitates of a GEV cell lysate with antisera that reacted with the erbB gene product had PI kinase activity, whereas no activity was detected in those of lysates of uninfected CEF cells. However, the activity was very weak compared with the total cellular activity. No difference in the PIP and DG kinase activities of immunoprecipitates of cell lysates of uninfected CEF cells and GEV cells was observed. These results suggest that the erbB gene product enhances inositol phospholipid metabolism and subsequent signal transduction, but that the erbB gene product is not involved directly in lipid kinases, although it is closely associated with lipid kinase.     

Calmodulin antagonism: a pharmacological approach for the inhibition of mediator release from mast cells

Several Ca2+ antagonists with either Ca2+-entry blocking or calmodulin (CaM) antagonistic properties and antiallergic drugs were investigated for their effects on mediator release from mast cells induced by different secretagogues (compound 48/80, concanavalin A, antigen-IgE and Ca2+ ionophore A23187) and for their ability to inhibit the function of CaM or phospholipid/Ca2+-dependent protein kinase (C-kinase). The effects of the different agents--with the only exception of cromolyn sodium--on histamine release elicited by compound 48/80 correlated well with their actions on two CaM-dependent enzymes whereas the activity of C-kinase was far less altered, or not altered at all. CaM antagonism of cloxacepride, picumast, oxatomide, fendiline and bepridil correlated not only with the inhibition of exocytosis evoked by compound 48/80 but also with that induced by A23187, concanavalin A and antigen-IgE. This indicates an action of these substances distal to the generation of the Ca2+ signal since the various secretagogues elevate the intracellular Ca2+ concentration by different mechanisms. However, prenylamine and thioridazine inhibited concanavalin A- and antigen-IgE-induced mediator release more potently and more effectively than that elicited by compound 48/80 or A23187. Therefore inhibition of allergic histamine release by these drugs may in part be dependent on an impairment of the Ca2+ signal. Since for each of two agents inhibition of histamine release (evoked by different releasers) parallels that of serotonin release it may be concluded that these mediators are secreted via the same mechanism. The results obtained with agents exhibiting different pharmacological properties but which share one common property, namely antagonism of CaM, strengthen the view that CaM is involved in exocytosis of mediators from mast cells.     

Rat mast cell phospholipase A2: activity toward exogenous phosphatidylserine and inhibiton by N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylserine

The presence of phospholipase A2 in intact rat peritoneal mast cells was investigated by using two synthetic radiolabeled phosphatidylserine (PS) substrates. Incubation of intact cells with 1-oleoyl-2-[3H]oleoyl-PS resulted in the release of a considerable quantity of [3H]oleic acid from the substrate. To establish that [3H]oleic acid release was mediated via direct enzymatic attack at the sn-2 position, we measured release of the [3H]serine moiety from the glycerol backbone of 1,2-dimyristoylphosphatidyl[3H]serine. This activity, which represents the combined actions of phospholipases C and D, was 10-fold lower than [3H]oleic acid release, indicating that neither of these enzymes is required for the release of the preponderance of [3H]oleic acid. These results establish the existence in intact rat mast cells of a phospholipase A2 active toward exogenous PS. Over the concentration range at which exogenous PS activates mast cell secretion, intact mast cells and broken cells possessed nearly equal levels of phospholipase A2 activity, and enzyme activity was 3--4-fold higher toward PS than phosphatidylcholine. Several agents were tested for their ability to inhibit phospholipase A2 in intact mast cells. Of the agents tested, an N-substituted derivative of PS previously identified as an inhibitor of mast cell secretion was shown to be a particularly potent and efficacious inhibitor of mast cell phospholipase A2. The concentration dependence of enzyme inhibition paralleled inhibition of histamine secretion, providing a strong positive correlation between the level of phospholipase A2 in mast cells and the capacity for secretion.     

Role of calcium in histamine release from rat mast cells activated by various secretagogues; intracellular calcium mobilization correlates with histamine release

Anti-IgE, con A or antigen caused an increase in the intracellular calcium concentration, [Ca2+]i, of mast cells. The increase occurred in two stages: a rapid initial rise caused by Ca-mobilization from intracellular Ca-stores and a second sustained rise caused by an influx of extracellular calcium (White, J.R., Pluznik, D.V., Ishizaka, K. & Ishizaka, T. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8193-8197). The rapid initial rise was followed by a release of histamine, which seemed to coincide with the second rise. A23187 and compound 48/80 induced a rapid initial rise in [Ca2+]i, followed by a gradual decrease in [Ca2+]i, GMCHA-OPhBut, a specific pH 7 tryptase inhibitor (Muramatu, M., Ito, T., Takei, M. & Endo, K. (1988) Biol. Chem. Hoppe-Seyler 369, 617-625), strongly inhibited both the initial and second rises of [Ca2+]i, as well as histamine release by these secretagogues, and its effects on the initial rise were closely correlated with those on histamine release. Addition of GMCHA-OPhBut immediately after the initial rise strongly inhibited the second rise, thereby decreasing the final [Ca2+]i. These results strongly suggested a possible involvement of pH 7 tryptase, not only in Ca-mobilization leading to the initial rise in [Ca2+]i, but also in the second rise. Trapping of extracellular calcium by 3mM EGTA decreased both the initial rise in [Ca2+]i and histamine secretion induced by anti-IgE or con A; the magnitude of this effect depended on the time between induction and EGTA addition. Histamine release was closely correlated with the initial rise in [Ca2+]i. Similar results were obtained with A23187, but even 5 min after the addition of EGTA an initial rise of [Ca2+]i could still be induced, and histamine (30% of total histamine) was still released. However, A23187 did not induce a rise in [Ca2+]i in mast cells which had been exhaustively washed with Tyrode/Hepes solution containing 3mM EGTA, followed by suspension in the same solution. Even at 20 min after depletion of the extracellular calcium, compound 48/80 still caused an initial rise in [Ca2+]i to above half the maximal value, and histamine secretion was even less affected. The above results indicated that the initial rise in [Ca2+]i, due to Ca-mobilization, correlates with the histamine release promoted by the secretagogues described. On the other hand, isoproterenol strongly induced histamine secretion with no change of [Ca2+]i, while EGTA treatment prior to isoproterenol stimulation had no effect on histamine release, indicating a different secretion mechanism.     

Characterization of the effects of phorbol esters on rat mast cell secretion

When applied to the skin, phorbol esters (PEs) elicit signs of acute inflammation, suggesting they may induce the release of mediators from mast cells. Therefore, we have studied the effects of PEs on purified rat peritoneal and thoracic mast cells both alone and in conjunction with the calcium ionophore, A23187, and various other secretagogues that interact with immunoglobulin E (e.g., anti-IgE and Con A) or other cell surface receptors, e.g., somatostatin and compd 48/80. PEs alone caused little or no release of histamine. However, the PE 12-O-tetradecanoylphorbol-13-acetate (TPA, 10 ng/ml) tremendously potentiated release induced by the calcium ionophore A23187, reducing the EC50 for A23187 from 832 ng/ml to 56 ng/ml. In the presence of suboptimal A23187 (50 ng/ml), only active tumor promoting PEs elicited histamine release. The EC50 values of the various active PEs were: TPA 5 ng/ml; 4 beta-PDD, 83 ng/ml; and 4-O-methyl-TPA, 807 ng/ml, with maximal histamine release ranging from 54 to 80%. TPA synergistically enhanced stimulation of histamine release by anti-IgE and Con A over the entire concentration-response range. In contrast, this synergism was absent when cells were stimulated with somatostatin and compd 48/80. Phorbol esters may act by increasing the activity of a calcium/phospholipid-dependent protein kinase (Ca/PL-PK). Mast cells do have Ca/PL-PK activity, and TPA in the presence of suboptimal A23187 induces protein phosphorylation comparable with other secretagogues. These results suggest that in the purified mast cell, PE-induced mediator release increases the sensitivity of release mechanisms for calcium, acts syngergistically with secretagogues interacting with IgE, and as suggested from structure-activity relationships, occurs via a specific mechanism of action perhaps involving the Ca/PL-PK.