Biomedical subjects

R Sagi-Eisenberg

Publications and source records attributed to R Sagi-Eisenberg.

At least 19 recordsLinked to original sources

PubMed2001

Gi-mediated activation of the Syk kinase by the receptor mimetic basic secretagogues of mast cells: role in mediating arachidonic acid/metabolites release.

Syk kinase is essential for FcepsilonRI-mediated signaling and release of inflammatory mediators from mast cells. We now show that activation of rat peritoneal mast cells by the nonimmunological, G(i)-mediated pathway also results in the activation of Syk. We show that compound 48/80 (c48/80), a receptor analogue that activates directly G proteins, activates Syk in a pertussis toxin-sensitive fashion. We further show that Syk activation by c48/80 is blocked by the protein kinase C inhibitor GF109203X, by the phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, by EGTA, and by the selective src-like kinase inhibitor PP1. These results suggest that in the nonimmunological, G(i)-mediated pathway, Syk is located downstream from phospholipase C and phosphatidylinositol 3-kinase. However, in common with the FcepsilonRI-mediated pathway, activation of Syk by c48/80 is dependent on a src-like protein tyrosine kinase. Finally, we show that in the nonimmunological pathway, Syk plays a central role in the release of arachidonic acid/eicosanoid metabolites, but not in the release of prestored mediators such as histamine.

Animals↗

PubMed2001

Synaptotagmin regulates mast cell functions.

Synaptotagmin(s) (Syts), are products of a gene family implicated in the control of Ca2+-dependent exocytosis. Mast cells, specialized secretory cells that release mediators of inflammatory and allergic reactions in a process of regulated exocytosis, express Syt homologues and SNAREs (Soluble NSF Attachment proteins Receptors), which together with Syt constitute the core complex which mediates exocytotic vesicle docking and fusion. Rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells, express the Syt homologues Syt II, Syt III and Syt V Expression of Syt I, the neuronal Ca2+ sensor, in the RBL cells, resulted in its targeting to secretory granules and in prominent potentiation and acceleration of Ca2+-dependent exocytosis. Syt II is localized to an amine-free lysosomal compartment, which is also subjected to regulated exocytosis. Lysosomal exocytosis is negatively regulated by Syt II: overexpression of Syt II inhibited Ca2+-triggered exocytosis of lysosomes, while suppression of Syt II expression markedly potentiated this release. These findings implicate Syt homologues as key regulators of mast cell function.

Animals↗

PubMed2000

Stimulation of Ca(2+)-dependent exocytosis and release of arachidonic acid in cultured mast cells (RBL-2H3) by quercetin.

Basic secretagogues, such as compound 48/80, stimulate secretion in rat peritoneal mast cells by directly activating the heterotrimeric G-protein Gi(3) (Aridor M, et al. Science 1993;262:1569-72). Cultured RBL-2H3 mast cells do not normally respond to basic secretagogues, but acquire such responsiveness upon prolonged exposure to the kinase inhibitor, quercetin, which also increases the cellular level of Gi(3) (Senyshyn J, Baumgartner RA, Beaven MA. J Immunol 1998;160:5136-44). Expression of a GTPase-deficient mutant of Galphai(3) in RBL-2H3 cells results in the stimulation of Ca(2+)-triggered exocytosis and release of arachidonic acid (AA) (Zussman A, Hermuet S, Sagi-Eisenberg R. Eur J Biochem 1998;258:144-6). Here we show that long-term incubation with quercetin markedly stimulates Ca(2+)-triggered exocytosis and release of AA from the RBL-2H3 cells. We further show that membranes derived from such quercetin-treated cells display a reduced GTPase, but not ATPase, activity. Taken together with our previous observations, these results further implicate Gi(3) as one of the cellular targets through which quercetin confers responsiveness towards the family of basic secretagogues.

Adenosine Triphosphatases↗

PubMed1999

Synaptotagmin II negatively regulates Ca2+-triggered exocytosis of lysosomes in mast cells.

Synaptotagmins (Syts) I and II are believed to act as Ca2+ sensors in the control of neurotransmission. Here we demonstrate that mast cells express Syt II in their lysosomal fraction. We further show that activation of mast cells by either aggregation of FcepsilonRI or by Ca2+ ionophores results in exocytosis of lysosomes, in addition to the well documented exocytosis of their secretory granules. Syt II directly regulates lysosomal exocytosis, whereby overexpression of Syt II inhibited Ca2+-triggered release of the lysosomal processed form of cathepsin D, whereas suppression of Syt II expression markedly potentiated this release. These findings provide evidence for a novel function of Syt II in negatively regulating Ca2+-triggered exocytosis of lysosomes, and suggest that Syt II-regulated secretion from lysosomes may play an important role in mast cell biology.

Animals↗

PubMed1999

Gi-mediated activation of mitogen-activated protein kinase (MAPK) pathway by receptor mimetic basic secretagogues of connective tissue-type mast cells: bifurcation of arachidonic acid-induced release upstream of MAPK.

The family of basic secretagogues of connective tissue mast cells act as receptor mimetic agents, which trigger exocytosis by directly activating G proteins. We now demonstrate that pertussis toxin (Ptx)-sensitive Gi proteins, activated by compound 48/80 (c48/80), a potent member of this family, also activate the p42/p44 MAP kinases (MAPKs). This activation was potentiated by the protein tyrosine phosphatase inhibitor vanadate, whereas the tyrphostin AG-18, a competitive inhibitor of protein tyrosine kinases (PTKs); the protein kinase C inhibitors K252a and GF109203X; the phosphatidylinositol-3-kinase (PI-3K) inhibitors wortmannin and LY294002; and EGTA have abolished this activation. These results suggest that c48/80 activated the p42/p44 MAPKs via a mechanism that involves PTKs, protein kinase C, phosphatidylinositol-3-kinase and Ca2+ as mediators. Protein tyrosine phosphorylation and activation of the p42/p44 MAPKs were closely correlated with stimulation of arachidonic acid (AA) release by c48/80 but not with histamine secretion. However, whereas PD98059, the inhibitor of the MAPK kinase has abrogated MAPK activation, this inhibitor failed to effect release of AA. We therefore conclude that by activating Ptx-sensitive Gi protein(s), the basic secretagogues of mast cells stimulate multiple signaling pathways, which diverge to regulate the production and release of the different inflammatory mediators. Whereas the signaling pathway responsible for triggering histamine release is PTK independent, the pathway responsible for the stimulation of AA release bifurcates downstream to PTKs but upstream to the activation of MAPKs.

Androstadienes↗

PubMed1998

Stimulation of Ca2+-dependent exocytosis and arachidonic acid release in cultured mast cells (RBL-2H3) by a GTPase-deficient mutant of G alpha i3.

Gi3, a member of the Gi family of heterotrimeric GTP-binding proteins, regulates vesicle trafficking along both the constitutive and regulated pathways. In mast cells, specialized secretory cells which secrete a variety of inflammatory mediators by regulated exocytosis, activation of Gi3 provides a sufficient signal for exocytosis [Aridor, M., Rajmilevich, G., Beaven, M. A. & Sagi-Eisenberg, R. (1993) Science 262, 1569-1572]. Such activation can be achieved in patch-clamped or streptolysin-O (SLO)-permeabilized mast cells by a combination of Ca2+ and nonhydrolyzable analogs of GTP. In contrast, Ca2+-activated exocytosis in intact cells is Gi3 independent. We show here that overexpression of a GTPase-deficient mutant (G alpha i3Q204L), but not of the wild-type form of G alpha i3, in rat basophilic leukemia cells (RBL-2H3), a tumor analog of mucosal mast cells, resulted in marked potentiation of exocytosis and release of arachidonic acid in intact cells activated by a Ca2+ ionophore alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, exocytosis and arachidonic acid release stimulated by aggregation of the cell surface receptors for immunoglobulin E (IgE) were unaffected. These results strongly suggest that the intracellular receptor, responsible for the activation of Gi3, is a low-affinity Ca2+-binding protein that can only be activated during Ca2+ ionophore stimulation. Moreover, these results also suggest that the propagation of the Ca2+-activated and Gi3-mediated signaling pathway requires the blocking of Gi3 GTPase activity. Finally, our results indicate that release of arachidonic acid is at least one of the downstream effectors of Gi3.

Amino Acid Sequence↗

PubMed1998

Ca2+-dependent exocytosis in mast cells is stimulated by the Ca2+ sensor, synaptotagmin I.

Mast cells secrete a variety of biologically active substances that mediate inflammatory responses. Synaptotagmin(s) (Syts) are a gene family of proteins that are implicated in the control of Ca2+-dependent exocytosis. In the present study, we investigated the possible occurrence and functional involvement of Syt in the control of mast cell exocytosis. Here, we demonstrate that both connective tissue type and mucosal-like mast cells express Syt-immunoreactive proteins, and that these proteins are localized almost exclusively to their secretory granules. Furthermore, expression of Syt I, the neuronal Ca2+ sensor, in rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells, resulted in prominent potentiation and acceleration of Ca2+-dependent exocytosis. Therefore, these findings implicate Syt as a Ca2+ sensor that mediates regulated secretion in mast cells to calcium ionophore.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

Basic secretagogues activate protein tyrosine phosphorylation and release of arachidonic acid in mast cells via a novel protein kinase C and phosphatidylinositol 3-kinase-dependent mechanism.

Mast cells play a central role in inflammatory and immediate-type allergic reactions. These granulated cells release by a process of regulated exocytosis a variety of biologically active substances which are either preformed (e.g. histamine), or synthesized de novo following activation [e. g. metabolites of arachidonic acid (AA) and multifunctional cytokines]. Exocytosis in mast cells is activated either in response to aggregation of the receptors for immunoglobulin E (FcepsilonRI) or by the direct activation of pertussis toxin-sensitive G-proteins by a class of receptor mimetic agents, collectively known as basic secretagogues of mast cells. In the present study we show that compound 48/80 (c48/80), a synthetic member of the class of basic secretagogues, stimulates protein tyrosine phosphorylation of a number of as yet unidentified cellular substrates. These phosphorylations were inhibited by the tyrphostin AG-18, by the phosphatidylinositol 3-kinase inhibitor wortmannin and by the protein kinase C inhibitors K252a and GF1 09203X. These inhibitors also inhibited the release of AA induced by c48/80 but had no effect on exocytosis. Taken together, our findings suggest that basic secretagogues induce protein tyrosine phosphorylation as part of their parallel multiple signaling pathways which are presumably mediated by more than one G-protein. Both protein kinase C and phosphatidylinositol 3-kinase serve as intermediates in this signaling pathway. The protein tyrosine kinase signaling pathway, which mediates the activation of AA release, does not contribute to secretion of the preformed mediators such as histamine, but it might largely contribute to the de novo production of inflammatory mediators like leukotrienes and prostaglandins.

Androstadienes↗

PubMed1994

Antigen and thapsigargin promote influx of Ca2+ in rat basophilic RBL-2H3 cells by ostensibly similar mechanisms that allow filling of inositol 1,4,5-trisphosphate-sensitive and mitochondrial Ca2+ stores.

In single, Fura 2-loaded RBL-2H3 cells, antigen and thapsigargin depleted the same intracellular pool of Ca2+ in the absence of external Ca2+; provision of external Ca2+ induced immediate increases in levels of free Ca2+ ([Ca2+]i). These increases were dependent on the presence of external Ca2+ and, presumably, on influx of Ca2+ across the cell membrane. Both stimulants enhanced intracellular accumulation of 45Ca2+ through ostensibly similar mechanisms because accumulation was blocked to similar extents by various multivalent cations or by depolarization with K+. Because thapsigargin blocked reuptake of Ca2+ into inositol 1,4,5-trisphosphate sensitive stores, uptake occurred independently of the refilling of these stores. Uptake was dependent instead on sequestration of 45Ca2+ in a pool of high capacity that was insensitive to thapsigargin, caffeine, GTP and inositol 1,4,5-trisphosphate but sensitive to ionomycin and mitochondrial inhibitors. The existence of an inositol 1,4,5-trisphosphate-insensitive pool was also apparent in permeabilized cells; at 0.1 microM [Ca2+]i, uptake of 45Ca2+ was largely confined (> 80%) to the inositol 1,4,5-trisphosphate-sensitive pool, but at 2 microM [Ca2+]i uptake was largely (> 60%) into the inositol 1,4,5-trisphosphate-insensitive pool. Provision of mitochondrial inhibitors along with thapsigargin to block uptake into both pools, did not impair the thapsigargin-induced increase in [Ca2+]i or influx of Ca2+, as indicated by changes in Fura 2 fluorescence, but did block the intracellular accumulation of 45Ca2+. The studies illustrate the utility of simultaneous measurements of [Ca2+]i and 45Ca2+ uptake for a full accounting of Ca2+ homoeostasis as exemplified by the ability to distinguish between influx and mitochondrial uptake of Ca2+.

Animals↗

PubMed1993

Activation of exocytosis by the heterotrimeric G protein Gi3.

Secretagogues of rat peritoneal mast cells, such as mastoparan and compound 48/80, induce mast cell exocytosis by activating directly the guanosine triphosphate-binding proteins that are required for exocytosis. The introduction of a synthetic peptide that corresponds to the carboxyl-terminal end sequence of G alpha i3 into the cells specifically blocked this secretion. Similar results were obtained when antibodies to this peptide were introduced. The G alpha i3 was located in both the Golgi and the plasma membrane, but only the latter source of G alpha i3 appeared to be essential for secretion. These results indicate that G alpha i3 functions to control regulated exocytosis in mast cells.

Amino Acid Sequence↗

PubMed1992

Synovial protein kinase C and its apparent insensitivity to interleukin-1.

Lapine synovial fibroblasts produce prostaglandin E2 (PGE2) and neutral metalloproteinases in response to phorbol 12-myristate 13-acetate (PMA), human recombinant interleukin-1 (hrIL-1) and, in an autocrine fashion, in response to partially purified preparations of their own cytokines known as cell-activating factors (CAF). Here we have examined the possible role of protein kinase C (PKC) in these responses. Whereas the 80-kDa substrate for PKC could not be detected in synovial fibroblasts, these cells contained a 35-kDa protein which fulfilled the criteria for qualifying as a specific substrate of PKC. Translocation assays based upon phosphorylation of the 35-kDa protein and Western blotting techniques allowed the movement of PKC from the cytosolic to the particulate fraction in response to PMA and CAF to be detected but not in response to 4 alpha-PMA or hrIL-1. Inhibitors of PKC suppressed synovial activation by PMA, partially blocked activation by CAF but had no effect on activation by hrIL-1. There thus appear to be PKC-dependent and PKC-independent routes to synovial cell activation. Our data suggest that IL-1 uses the latter, while CAF contains cytokines which utilize both routes.

Animals↗

PubMed1991

Purification of p100, a protein antigenically related to the signal transducing G proteins Gt and Gi. Evidence for an adaptin-like protein.

A 100-kDa protein, termed p100, cross-reacts with antisera raised against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of the retinal G protein Gt. p100 is abundantly expressed in liver and, on subcellular fractionation of rat liver homogenates, is distributed between the cytosolic and microsome fractions (Traub, L. M., Evans, W. H., and Sagi-Eisenberg, R. (1990) Biochem. J. 272, 453-458; Udrisar, D., and Rodbell, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6321-6325). We have now purified p100 to near-homogeneity from rat liver microsomes. The protein was purified approximately 500-fold by ATP extraction followed by a series of four chromatographic steps. Similar to partially purified p100, on two-dimensional electrophoresis, the final preparation contained a major series of five immunoreactive 100-kDa charge isoforms. Partial amino terminus amino acid sequencing of the purified protein revealed that p100 is a previously unidentified protein. Further analysis of the soluble form of p100 showed the protein migrated with an apparent molecular weight of approximately 110,000 on gel filtration, indicating that the soluble protein occurs as a monomeric polypeptide. The soluble form of p100 was also partially purified from rat liver cytosol and amino acid sequencing yielded the same amino-terminal sequence as obtained from the microsome-associated form. The amino-terminal sequence of p100 exhibits significant similarity to the deduced amino-terminal amino acid sequences of both alpha- and gamma-adaptins. Using the amino-terminal sequence from p100, we have raised antipeptide polyclonal antisera. The antisera reacted specifically with the purified 100-kDa protein on immunoblots. With the purified protein and specific antisera now available, it will be possible to explore the physiological role of p100.

Amino Acid Sequence↗

PubMed1991

Characterization of the interaction between p100, a novel G-protein-related protein, and rat liver endosomes.

p100 is a recently identified 100 kDa protein which shares a putative receptor-binding sequence with the signal transducing G-proteins Gt and Gi. In liver, p100 immunoreactivity is distributed between the cytosolic and the microsomal fractions [Traub, Evans & Sagi-Eisenberg (1990) Biochem. J. 272, 453-458; Udrisar & Rodbell (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6321-6325]. More specifically, we have localized the membrane-associated form of p100 to an endosomal subfraction of rat liver microsomes. In this study we have investigated the nature of the interaction between p100 and microsomal membranes. p100 was located on the cytoplasmic surface of the microsomal vesicles, and could be released by treatment with 0.5 M-NaCl or 0.5 M-Tris/HCl, pH 7.0. However, p100 was not released by non-ionic detergents, such as Triton X-100. Binding of p100 to the membrane was reversible, as both membrane-released and cytosolic p100 could re-bind stripped (Tris-washed) microsomes. Soluble p100 could not, however, bind to untreated microsomes. Binding to stripped microsomes approached saturation and was inhibited by up to 60% by either heat treatment or mild trypsin treatment of the vesicles. This implies that the interaction between p100 and the microsomal vesicles involves the direct binding of p100 to vesicular proteins. This binding was regulated by both adenine and guanine nucleotides. As p100 contains a region similar to the C-terminal decapeptide of alpha i, (the alpha-subunit of Gi) and has a localization that is restricted to an endosomal subfraction, we propose that cytosolic p100 may bind to cytoplasmically exposed domains of internalized receptors. Thus, like the adaptins, p100 may be involved in the process of sorting and receptor trafficking through the endosomal compartment of the cells.

Animals↗

PubMed1991

Interleukin-1 and synovial protein kinase C: identification of a novel, 35 kDa cytosolic substrate.

We have been examining the role of protein kinase C (PKC) in synovial cell activation in response to interleukin-1 (IL-1). Attempts to measure PKC in soluble extracts of synovial fibroblasts by standard techniques failed. Western blotting with anti-PKC antibodies detected only a low level of PKC in synovial cells compared to rat basophilic leukemia cells and crude brain extracts. However, synovial PKC could be detected by measuring the Ca(2+)- and phospholipid-dependent phosphorylation of endogenous substrates. In this way, a 35 kDa protein was identified as the major endogenous cytosolic substrate for PKC. Treatment of synoviocytes with phorbol myristate acetate (PMA) strongly induced the synthesis of neutral metalloproteinases (NPs) and prostaglandin E2 (PGE2). Both Western blotting and assays based upon phosphorylation of the 35 kDa protein confirmed translocation of PKC from the cytosol in response to PMA. Although IL-1 induced the NPs and PGE2, it did so without detectable translocation of PKC. There thus appear to be PKC-dependent and PKC-independent routes of synovial cell activation. Our data suggest that IL-1 uses the latter.

Animals↗

PubMed1990

A novel 100 kDa protein, localized to receptor-enriched endosomes, is immunologically related to the signal-transducing guanine-nucleotide-binding proteins Gt and Gi.

Antisera raised against the C-terminus decapeptide of the alpha-subunit of the retinal guanine-nucleotide-binding protein (G-protein) transducing (Gt) cross-reacted with the alpha-subunit of the inhibitory G-protein Gi. The same antisera also reacted with a 100 kDa protein (p100) found in rat liver homogenates. The immunoreactivity of both Gt and p100 was specifically inhibited by the immunizing peptide with similar dose-dependencies [concn. causing 50% inhibition (IC50) = 300 ng/ml]. This similarity in inhibition profiles implies that p100 contains within its structure the C-terminal sequence shared by both alpha t and alpha i. Tissue distribution studies demonstrated that p100 was particularly enriched in the liver and kidney, but was also present in other rat tissues, as well as in a number of cell lines tested. In the liver, p100 was found in both the soluble and membrane fractions. The membrane-associated form of p100 was specifically localized to an endosomal fraction (termed D-R), previously shown to be a ligand-free but receptor-enriched subfraction of liver endosomal vesicles. Two-dimensional gel electrophoresis revealed that both the cytosolic and membrane-bound forms of p100 occurred as a series of 100 kDa polypeptides with considerable charge heterogeneity (pI 6-7). Because the C-terminus domains of both alpha t and alpha i facilitate their association with their respective receptors, this region has been functionally assigned as the receptor binding site. Therefore the presence of an immunologically similar region within p100, together with its localization to the receptor-rich endocytic vesicles, suggests that p100 may be a receptor binding protein involved in receptor trafficking.

Animals↗

PubMed1990

A combination of H2O2 and vanadate concomitantly stimulates protein tyrosine phosphorylation and polyphosphoinositide breakdown in different cell lines.

Treatment of four cell lines [rat hepatoma (Fao), murine muscle (BC3H-1), Chinese hamster ovary (CHO), and rat basophilic leukemia (RBL)] with a combination of 3 mM H2O2 and 1 mM sodium orthovanadate markedly stimulates protein tyrosine phosphorylation, which is accompanied by a dramatic increase (5-15-fold) in inositol phosphate (InsP) formation. H2O2/vanadate stimulate best formation of inositol triphosphate while their effects on the mono and di derivatives are more moderate. In the presence of 3 mM H2O2, both protein tyrosine phosphorylation and InsP formation are highly correlated and manifest an identical dose-response relationship for vanadate. Half-maximal and maximal effects are obtained at 30 and 100 microM, respectively. This stimulatory effect of H2O2/vanadate is not mimicked by other oxidants such as spermine, spermidine, KMnO4, and vitamin K3. In RBL cells, the kinetics of inositol triphosphate formation correlate with tyrosine phosphorylation of a 67-kDa protein, while tyrosine phosphorylation of a 55-kDa protein is closely correlated with both inositol monophosphate formation and serotonin secretion from these cells. Taken together, these results suggest a causal relationship between tyrosine phosphorylation triggered in a nonhormonal manner and polyphosphoinositide breakdown. Furthermore, these results implicate protein tyrosine phosphorylation in playing a role in the stimulus-secretion coupling in RBL cells.

Animals↗

PubMed1990

Differential down-regulation of protein kinase C selectively affects IgE-dependent exocytosis and inositol trisphosphate formation.

Short-term treatment of rat basophilic leukaemia (RBL-2H3) cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activates protein kinase C (PKC) and results in the inhibition of the IgE-dependent formation of inositol phosphates, but in the potentiation of serotonin secretion. Long-term treatment with TPA, which depletes the cells of their endogenous PKC, eliminates both Ca2(+)-ionophore- and TPA- as well as IgE-dependent secretion, but it potentiates by 1.7-fold IgE-induced inositol phosphate formation. Taken together, these observations strongly suggest that the dual actions of TPA on IgE-dependent responses are both mediated by PKC. The opposing effects of TPA are differentially down-regulated. Following TPA treatment, the rate by which the cells lose their ability to undergo exocytosis is faster than the rate at which inhibition of inositol phosphates formation is relieved and their production potentiated. In addition, both processes show different sensitivities to inhibitors of PKC action. Whereas IgE-dependent secretion is completely blocked by the PKC inhibitors K252a, H-7 and sphingosine [concns. causing 50% inhibition (IC50 values) = 25 ng/ml 80 microns and 30 microns respectively], these inhibitors do not relieve inhibition of inositol phosphate formation by TPA, nor do they potentiate this response. These results may imply that the bidirectional control exerted by PKC on IgE-dependent responses is mediated by its different isoenzymes.

Animals↗