Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells

The cytoskeletal changes that alter cellular morphogenesis and motility depend upon a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. The Rho family of GTP binding proteins are important upstream mediators of cytoskeletal organization. Gem and Rad are members of another family of small GTP binding proteins (the Rad, Gem, and Kir family) for which biochemical functions have been mostly unknown. Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) alpha and beta. Gem binds ROKbeta independently of RhoA in the ROKbeta coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKbeta-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKbeta. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKbeta- and Rad opposed ROKalpha-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKbeta containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKbeta is required for the effects observed. In epithelial or fibroblastic cells, Gem or Rad expression resulted in stress fiber and focal adhesion disassembly. In addition, Gem reverted the anchorage-independent growth and invasiveness of Dbl-transformed fibroblasts. These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK.     

IgE-mediated histamine release in rat basophilic leukemia cells: receptor activation, phospholipid methylation, Ca2+ flux, and release of arachidonic acid

Stimulation of IgE receptors on rat basophilic leukemia cells causes a transient rise and fall of methylated phopholipids, Ca²⁺ influx, and release of arachidonic acid previously incorporated into phosphatidylcholine and liberation of histamine. Inhibition of phospholipid methylation by methyltransferase inhibitors, 3-deazaadenosine and homocysteine thiolactone, almost completely blocks the influx of Ca²⁺, and release of arachidonic acid and histamine. Stimulation of immunoglobulin E receptors by antigen releases only [¹⁴C]arachidonic acid but not [¹⁴C]linoleic acid, [¹⁴C]oleic acid and [¹⁴C]stearic acid, all of which were previously incorporated into phospholipids. [¹⁴C]Arachidonate was found to be incorporated mainly into phosphatidylcholine. The phosphatidycholine rich in arachidonate appeared to be synthesized to a considerable extent by the transmethylation pathway. These findings suggest that in rat basophilic leukemia cells, immunoglobulin E receptors, phospholipid methyltransferases, Ca²⁺ ion channel, and phospholipase(s) that cause release of arachidonic acid and the discharge of histamine are associated.     

Prolactin secretion in permeable GH3 pituitary cells is stimulated by Ca2+ and protein kinase C activators

We have used GH3 cells permeabilized by electric field discharge to examine the effects of Ca2+ and protein kinase C activators (phorbol ester and diacylglycerol) on prolactin (PRL) release. Ca2+ was found to stimulate PRL release approximately 4 fold at 3 microM Ca2+ with a half-maximal response at approximately .5 microM estimated free Ca2+. 12-O-tetradecanoyl phorbol-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol stimulated PRL release throughout a range of Ca2+ concentrations (1 nM -3 microM), but stimulation was greater at higher Ca2+ concentrations (.1 microM to 1 microM). Both agents decreased by 1.8 fold the apparent [Ca2+] at which half-maximal stimulation of secretion occurred. Quin 2 was used to measure the free [Ca2+] of intact and permeable cells; PRL secretion at a free [Ca2+] corresponding to resting cytoplasmic [Ca2+] was 10% of maximal, while secretion at the [Ca2+] corresponding to the Ca2+ spike induced by thyrotropin-releasing hormone was approximately 25% of maximal.     

Arachidonic acid and diacylglycerol act synergistically to activate protein kinase C in vitro and in vivo

Using a well-defined model membrane bilayer system, incorporation of both lipid second messengers, 1,2-diacylglycerol and arachidonic acid, at submaximal activating concentrations, resulted in a synergistic activation of protein kinase C in a Ca2+/phosphatidylserine-dependent manner as measured by monitoring phosphorylation of phosphoprotein substrates. The arachidonic acid appears to modulate membrane properties both at the hydrocarbon core and the membrane surface increasing the availability of the diacylglycerol which can bind to and subsequently activate the enzyme. Co-application of these two lipid activators to the Hermissenda photoreceptor reduced K+ channel conductance in a synergistic manner via a PKC-dependent pathway. Thus, these in vivo and in vitro studies suggest that the membrane bilayer properties of these PKC lipid activators interact to specifically regulate the cellular lipid microenvironment resulting in PKC activation.     

Regulation of the antigen-induced F-actin response in rat basophilic leukemia cells by protein kinase C

Multivalent antigen that is capable of binding to and crosslinking the IgE receptors on rat basophilic leukemia (RBL) cells, induces a rapid and sustained rise in the content of filamentous actin. This reorganization of the actin may be responsible for changes in cellular morphology during the degranulation process. The antigen-stimulated polymerization of actin can be blocked in a dose-dependent manner by protein kinase inhibitors which also block degranulation. Conversely, reagents such as PMA, 1,2-dioctanoyl-sn-glycerol (diC8), and 1-oleoyl-2-acetyl-glycerol (OAG) which stimulate protein kinase C (PKC) also activate the rise in F-actin, although they have no effect on degranulation by themselves. The actin response which can be stimulated by the PKC activators can also be blocked by protein kinase inhibitors indicating that the PMA- and OAG-induced response is probably through activation of a protein kinase. Depletion of PKC activity through long term (20 h) exposure of RBL cells to PMA, also inhibited the F-actin response when the cells were stimulated with either multivalent antigen or OAG. External Ca++, which is an absolute requirement for degranulation, is not necessary for the rise in F-actin, but may modulate the response. Furthermore, ionomycin, which induces a large Ca++ influx, does not stimulate the F-actin increase even at doses that cause degranulation. These results suggest that activation of a protein kinase, such as PKC, may be responsible for signaling the polymerization of actin in RBL cells and that a rise in intracellular Ca++ is neither necessary nor sufficient for this response.     

Synergistic release of arachidonic acid from platelets by activators of protein kinase C and Ca2+ ionophores. Evidence for the role of protein phosphorylation in the activation of phospholipase A2 and independence from the Na+/H+ exchanger

The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ ionophores A23187 and ionomycin. In contrast, 4 alpha-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2. Synergism between PMA and A23187 is not affected by inactivation of the Na+/H+ exchanger with dimethylamiloride. The time course and dose-response for the effect of PMA at 23 degrees C closely correlate with the phosphorylation of a set of relatively "slowly" phosphorylated proteins (P20, P35, P41, P60), but not the rapidly phosphorylated P47 protein. P20 is myosin light chain, and P41 is probably Gi alpha, but the other proteins have not been positively identified. Depletion of metabolic ATP stores by antimycin A plus 2-deoxyglucose abolishes both protein phorphorylation and the potentiation of arachidonate release by PMA, but does not prevent fatty acid release by the ionophores. Similarly, the kinase C inhibitors H-7 and staurosporine produce, respectively, partial and complete inhibition of PMA-potentiated arachidonic acid release and protein phosphorylation, without affecting the direct response to ionophores. These results indicate that protein phosphorylation, mediated by kinase C, promotes the phospholipase A2 dependent release of arachidonic acid in platelets when intracellular Ca2+ is elevated by Ca2+ ionophores.     

Characterization of Ca2+-activated, phospholipid-dependent protein kinase C/protein kinase M in guinea pig peritoneal exudate macrophages

Protein kinase C (C-kinase) is shown to be present in the cytosolic and particulate fractions of mineral oil induced peritoneal macrophages of guinea pigs. By omission or use of a high concentration of leupeptin, three forms of the enzyme were obtained: stimulant/Ca2+/phospholipid-dependent C-kinase, eluted from DEAE 52 cellulose at 0.08-0.16 M NaCl; stimulant/Ca2+/phospholipid-independent protein kinase M (M-kinase), and Ca2+-inhibited & stimulant/phospholipid-dependent form of protein kinase, both eluted from DEAE 52 cellulose at 0.18-0.22 M NaCl. Phorbol ester or 1,2-diacylglycerol were used as stimulants. It is suggested that Ca2+-inhibited & stimulant/phospholipid-dependent protein kinase represents the in vivo form of the M-kinase in intact cells.     

Identity of common phosphoprotein substrates stimulated by interleukin 2 and diacylglycerol suggests a role of protein kinase C for IL 2 signal transduction

Interleukin 2 (IL 2) is a polypeptide growth factor essential for the proliferation and differentiation of T lymphocytes, large granulocytic lymphocytes, and, potentially, cells of the antibody-producing lineage, B lymphocytes. Many of the biological properties of IL 2 may be mimicked or potentiated by a potent class of tumor promoters, phorbol esters. Phorbol esters have recently been shown to associate with and activate a unique phospholipid/Ca2+-dependent phosphotransferase, protein kinase C (PK-C). Utilizing two-dimensional gel electrophoresis, we have compared the IL 2 and diacylglycerol-induced protein phosphorylation patterns of several IL 2-dependent murine cell lines. Both IL 2 and synthetic diacylglycerol, 1-oleyl-2-acetylglycerol (OAG), stimulated phosphorylation of a number of protein substrates in intact cells compared to unstimulated controls. Three groups of substrates were identified; the first showed increased phosphorylation following stimulation with either IL 2 or OAG, while the second and third groups showed increased phosphorylation following stimulation with IL 2 but not OAG, and with OAG but not IL 2, respectively. Here, we characterize the kinetics of phosphorylation of one cellular substrate, p68, which appears to be phosphorylated in response to direct activators of PK-C or lymphoid or myeloid growth factors in their respective lineage cell lines. The observation that IL 2 also stimulates a unique series of phosphoproteins in addition to those induced by direct PK-C activators suggests that IL 2 may initiate additional protein kinase activities, unrelated to PK-C, which may also be critical for the ligand-receptor signal transduction process regulating growth and gene expression.     

Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C

We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.     

Arachidonic acid release in rat peritoneal mast cells stimulated with antigen, ionophore A23187, and compound 48/80

Rat peritoneal mast cells respond to various types of secretagogues, such as antigen (receptor-mediated), A23187 (calcium mobilizing), and compound 48/80 (membrane perturbing), and release arachidonic acid from membrane phospholipids prelabeled with [3H]arachidonate. The rate of arachidonic acid liberation varied from one stimulant to the other. Ionophore A23187 (0.1 micrograms/ml) appeared to be most potent in releasing arachidonate among the three stimulants at which doses each secretagogue caused almost equivalent histamine secretion. However, upon stimulation with these three secretagogues, the radioactivity of phosphatidylcholine (PC) was markedly reduced with a concomitant increase of arachidonate radioactivity. Hydrolysis of PC by phospholipase A2 is likely to be the major route of arachidonic acid liberation in either IgE-mediated or non-IgE activation in mast cells.     

Modulation of Ca2+ mobilization by protein kinase C in rat submandibular acinar cells

The effects of protein kinase C (PKC) activation and inhibition on the inositol 1,4,5-trisphosphate (IP3) and cytosolic Ca2+ ([Ca2+]i) responses of rat submandibular acinar cells were investigated. IP3 formation in response to acetylcholine (ACh) was not affected by the PKC activator phorbol 12-myristate 13-acetate (PMA), nor by the PKC inhibitor calphostin C (CaC). The ACh-elicited initial increase in [Ca2+]i in the absence of extracellular Ca2+ was not changed by short-term (0.5 min) exposure to PMA, but significantly reduced by long-term (30 min) exposure to PMA, and also by pre-exposure to the PKC inhibitors CaC and chelerythrine chloride (ChC). After ACh stimulation, subsequent exposure to ionomycin caused a significantly (258%) larger [Ca2+]i increase in CaC-treated cells than in control cells. However, pre-exposure to CaC for 30 min did not alter the Ca2+ release induced by ionomycin alone. These results suggest that the reduction of the initial [Ca2+]i increase is due to an inhibition of the Ca2+ release mechanism and not to store shrinkage. The thapsigargin (TG)-induced increase in [Ca2+]i was significantly reduced by short-term (0.5 min), but not by long-term (30 min) exposure to PMA, nor by pre-exposure to ChC or CaC. Subsequent exposure to ionomycin after TG resulted in a significantly (70%) larger [Ca2+]i increase in PMA-treated cells than in control cells, suggesting that activation of PKC slows down the Ca2+ efflux or passive leak seen in the presence of TG. Taken together, these results indicate that inhibition of PKC reduces the IP3-induced Ca2+ release and activation of PKC reduces the Ca2+ efflux seen after inhibition of the endoplasmic Ca2+-ATPase in submandibular acinar cells.     

Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks

In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.     

Modulation by phorbol 12-myristate 13-acetate of arachidonic acid release from rat basophilic leukemia cells stimulated with A23187

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.     

Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells

IgE-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells results in the secretion of histamine. Myosin immunoprecipitated from these cells shows an increase in the amount of radioactive phosphate incorporated into its heavy (200 kDa) and light (20 kDa) chains. In unstimulated cells two-dimensional mapping of tryptic peptides of the myosin light chain reveals one phosphopeptide containing the serine residue phosphorylated by myosin light chain kinase. Following stimulation a second phosphopeptide appears containing a serine residue phosphorylated by protein kinase C. Tryptic phosphopeptide maps derived from myosin heavy chains show that unstimulated cells contain three major phosphopeptides. Following stimulation a new tryptic phosphopeptide appears containing a serine site phosphorylated by protein kinase C. The stoichiometry of phosphorylation of the myosin light and heavy chains was determined before and after antigenic stimulation. Before stimulation, myosin light chains contained 0.4 mol of phosphate/mol of light chain all confined to a serine not phosphorylated by protein kinase C. Cells that secreted 44% of their total histamine in 10 min exhibited an increase in phosphate content at sites phosphorylated by protein kinase C from 0 mol of phosphate/mol of myosin subunit to 0.7 mol of phosphate/mol of light chain and to 1 mol of phosphate/mol of heavy chain. When RBL-2H3 cells were made permeable with streptolysin O they still showed a qualitatively similar pattern of secretion and phosphorylation. Our results show that the time course of histamine secretion from stimulated RBL-2H3 cells parallels that of myosin heavy and light chain phosphorylation by protein kinase C.     

Translational suppression by Ca2+ ionophores: Reversibility and roles of Ca2+ mobilization, Ca2+ influx, and nucleotide depletion

The divalent cation selective ionophores A23187 and ionomycin were compared for their effects on the Ca²⁺ contents, nucleotide contents, and protein synthetic rates of several types of cultured cells. Both ionophores reduced amino acid incorporation by approximately 85% at low concentrations (50–300 nmol/L) in cultured mammalian cells without reducing ATP or GTP contents. At these concentrations A23187 and ionomycin each promoted substantial Ca²⁺ efflux, whereas at higher concentrations a large influx of the cation was observed. Ca²⁺ influx occurred at lower ionophore concentrations and to greater extents in C6 glioma and P3X63Ag8 myeloma than in GH₃ pituitary cells. The ATP and GTP contents of the cells and their ability to adhere to growth surfaces declined sharply at ionophore concentrations producing increased Ca²⁺ influx. Prominent reductions of nucleotide contents occurred in EGTA-containing media that were further accentuated by extracellular Ca²⁺. Ionomycin produced more Ca²⁺ influx and nucleotide decline than comparable concentrations of A23187. The inhibition of amino acid incorporation and mobilization of cell-associated Ca²⁺ by ionomycin were readily reversed in GH₃ cells by fatty acid-free bovine serum albumin, whereas the effects of A23187 were only partially reversed. Amino acid incorporation was further suppressed by ionophore concentrations depleting nucleotide contents. Mitochondrial uncouplers potentiated Ca²⁺ accumulation in response to both ionophores. At cytotoxic concentrations Lubrol PX abolished protein synthesis but did not cause Ca²⁺ influx. Nucleotide depletion at high ionophore concentrations is proposed to result from increased plasmalemmal Ca²⁺-ATPase activity and dissipation of mitochondrial proton gradients and to cause intracellular Ca²⁺ accumulation. Increased Ca²⁺ contents in response to Ca²⁺ ionophores are proposed as an indicator of ionophore-induced cytotoxicity.Abbreviations BSA bovine serum albumin - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - PKR double-stranded RNA-regulated protein kinase - ER endoplasmic reticulum - eIF eukaryotic initiation factor     

Phospholipase activation in the IgE-mediated and Ca2+ ionophore A23187-induced release of histamine from rat basophilic leukemia cells

The importance of phospholipase(s) activation in the IgE-mediated and ionophoreinduced histamine release from the rat basophilic leukemia cell line has been examined. The activation of phospholipase(s) as measured by [¹⁴C]arachidonic acid release and the release of histamine both required Ca²⁺ and were temporally parallel. Inhibition of phospholipase(s) activity by the inhibitors mepacrine and α-parabromoacetophenone also correlated with the inhibition of histamine release. [¹⁴C]Arachidonic acid released by the phospholipase(s) was mainly metabolized to prostaglandin D₂. The inhibition of the cyclooxygenase pathway by indomethacin did not affect histamine release. 5,8,11,14-Eicosatetraynoic acid inhibited both histamine and [¹⁴C]arachidonic acid release suggesting an effect not only on the cyclooxygenase and lipoxygenase pathways but also on the phospholipase(s). These results suggest that activation of phospholipase appears to be necessary for histamine release in the rat bosophilic leukemia cells.     

Regulation of erythrocyte Ca2+ pump activity by protein kinase C

Using either inside-out vesicles (IOV) prepared from human erythrocytes or purified Ca2+-ATPase from the same source, the effects of protein kinase C (Ca2+/phospholipid-dependent enzyme) on Ca2+ transport and Ca2+-ATPase activity were measured. Incubation of IOV with protein kinase C in the presence, but not absence, of either 12-O-tetradecanoylphorbol-13-acetate or diolein led to a Ca2+-dependent stimulation of ATP-dependent calcium uptake. The effect was a 5-7-fold increase of Vmax without a significant change in the apparent Km for Ca2+. By comparison, the effect of calmodulin was a 14-fold stimulation of Vmax and a 4-fold reduction in apparent Km. The effect of protein kinase C and calmodulin on Ca2+ uptake were nearly additive. Stimulation of IOV Ca2+ transport by protein kinase C was entirely reversible by treatment of activated IOV with alkaline phosphatase. Incubation of purified Ca2+-ATPase with protein kinase C in the presence of 12-O-tetradecanoylphorbol-13-acetate or diolein led to a stimulation of Ca2+-dependent ATPase activity. These results indicate that protein kinase C stimulates the activity of the plasma membrane Ca2+ pump by a direct effect on the pump protein.     

A novel action of glucocorticosteroid in inhibition of leukotriene C4 production by rat basophilic leukemia cells: suppression of the elevation of cytosolic free Ca2+ induced by antigen

Rat basophilic leukemia (RBL-2H3) cells serve as a model to examine the role of elevated internal Ca2+ concentration ([Ca2+]i), following antigen (DNP10BSA)-induced stimulation of leukotriene C4 (LTC4) formation. A novel action of hydrocortisone (HC), to reduce increased [Ca2+]i and consequently inhibit LTC4 formation is assessed. Half-maximal time for elevation of [Ca2+]i induced by antigen was less than 1 min, and maximal elevation of [Ca2+]i (3-fold increase) was reached within 2-3 min. This high [Ca2+]i level waned gradually by 27% during 20 min of incubation. For induction of LTC4 formation, however, there was a refractory period of about 2 min, and half-maximal elevation was at 11 min. Following pretreatment with HC, the antigen-stimulated increase in [Ca2+]i was stunted by 41% at 2-3 min and by 73% at 20 min. LTC4 formation was almost abolished. There was a lag period of at least 2 h to observe any inhibition in both parameters, and the maximal inhibition was about 4 h. Cycloheximide, and receptor antagonist to glucocorticosteroid (RU486) completely prevented the inhibitory effects of HC on elevated [Ca2+]i and LTC4 formation. Estradiol and aldosterone (each at 2.10(-6) M) were virtually inactive, while another glucocorticosteroid, dexamethasone (2.10(-7) M) markedly suppressed antigen induction in both parameters. It is proposed that the inhibitory effect of HC on the formation of LTC4 could be attributed mainly to its ability to reduce elevated [Ca2+]i.