A population-based epidemiological study of neuromyelitis optica spectrum disorder in Hungary.

BACKGROUND AND PURPOSE: The goal of this study was to determine the prevalence and incidence of neuromyelitis optica spectrum disorder (NMOSD) in Hungary based on the 2015 International Panel of NMO Diagnosis (IPND) criteria. METHODS: A retrospective population-based cohort study was conducted of 6.4 million Hungarians (age ≥ 16 years) between 1 January 2006 and 31 December 2016. Possible NMOSD patients were selected via multistage re-evaluation from multiple sources. Crude and sex- and serostatus-specific prevalence (per 100 000 persons) and incidence rates (per 1 000 000 person-years) from 2006 to 2015 were estimated and age-adjusted rates were determined. RESULTS: Of 2262 study candidates, 154 NMOSD patients (age ≥ 16 years) with onset until 31 December 2016 were identified based on 2015 IPND criteria. The prevalence analysis on 1 January 2016 included 123 NMOSD living cases, resulting in a prevalence of 1.91 [95% confidence interval (CI) 1.52-2.28] per 100 000 persons. The 101 incident cases emerging from the observed 76 394 288 person-years provided an incidence rate of 1.32 (95% CI 1.08-1.61) per 1 000 000 person-years. Age-adjusted prevalence was 1.87 (95% CI 1.56-2.23) per 100 000 persons and incidence was 1.20 (95% CI 0.98-1.46) per 1 000 000 person-years. CONCLUSIONS: In this first report of a large population-based epidemiological study from an Eastern European Caucasian population using robust case validation, a greater prevalence and incidence of NMOSD was found compared to previous large studies in Caucasian populations.

Cuprizone Administration Alters the Iron Metabolism in the Mouse Model of Multiple Sclerosis.

Cuprizone (CZ) is a widely used copper chelating agent to develop non-autoimmune animal model of multiple sclerosis, characterized by demyelination of the corpus callosum (CC) and other brain regions. The exact mechanisms of CZ action are still arguable, but it seems that the only affected cells are the mature oligodendrocytes, possibly via metabolic disturbances caused by copper deficiency. During the pathogenesis of multiple sclerosis, high amount of deposited iron can be found throughout the demyelinated areas of the brain in the form of extracellular iron deposits and intracellularly accumulated iron in microglia. In the present study, we used the accepted experimental model of 0.2% CZ-containing diet with standard iron concentration to induce demyelination in the brain of C57BL/6 mice. Our aim was to examine the changes of iron homeostasis in the CC and as a part of the systemic iron regulation, in the liver. Our data showed that CZ treatment changed the iron metabolism of both tissues; however, it had more impact on the liver. Besides the alterations in the expressions of iron storage and import proteins, we detected reduced serum iron concentration and iron stores in the liver, together with elevated hepcidin levels and feasible disturbances in the Fe-S cluster biosynthesis. Our results revealed that the CZ-containing diet influences the systemic iron metabolism in mice, particularly the iron homeostasis of the liver. This inadequate systemic iron regulation may affect the iron homeostasis of the brain, eventually indicating a relationship among CZ treatment, iron metabolism, and neurodegeneration.

Examination of the interaction of different lighting conditions and chronic mild stress in animal model.

We examined the effects of different shift work schedules and chronic mild stress (CMS) on mood using animal model. The most common international shift work schedules in nursing were applied by three groups of Wistar-rats and a control group with normal light-dark cycle. One subgroup from each group was subjected to CMS. Levels of anxiety and emotional life were evaluated in light-dark box. Differences between the groups according to independent and dependent variables were examined with one- and two-way analysis of variance, with a significance level defined at p < 0.05. Interaction of lighting regimen and CMS was proved to be significant according to time spent in the light compartment and the average number of changes between the light and dark compartments. Results of our examination confirm that the changes of lighting conditions evocate anxiety more prominently than CMS. No significant differences were found between the results of the low rotating group and the control group, supposing that this schedule is the least harmful to health. Our results on the association between the use of lighting regimens and the level of CMS provide evidence that the fast rotating shift work schedule puts the heaviest load on the organism of animals.

Distribution of oligodendrocyte loss and mitochondrial toxicity in the cuprizone-induced experimental demyelination model.

Cuprizone is a copper-chelating mitochondrial toxin that causes oligodendrocyte apoptosis and demyelination preferentially in the corpus callosum (CC) and the superior cerebellar peduncles, but not in the spinal cord (SC) of C57BL/6 mice. Here we aimed to determine the activities of copper-containing enzymes in correlation with the distribution of demyelination during exposure to cuprizone. The study revealed mitochondrial complex IV and superoxide dismutase activity alterations in both the pathology-affected CC and the non-affected SC. This observation raises the possibility that regionally different subcellular molecular interactions lead to the selective oligodendrocyte loss induced by the nonselective mitochondrial toxin, cuprizone.

Opposite roles of protein kinase C isoforms in proliferation, differentiation, apoptosis, and tumorigenicity of human HaCaT keratinocytes.

We have previously shown that the protein kinase C (PKC) system plays a pivotal role in regulation of proliferation and differentiation of the human keratinocyte line HaCaT which is often used to assess processes of immortalization, transformation, and tumorigenesis in human skin. In this paper, using pharmacological and molecular biology approaches, we investigated the isoform-specific roles of certain PKC isoenzymes (conventional cPKCalpha and beta; novel nPKCdelta and epsilon) in the regulation of various keratinocyte functions. cPKCalpha and nPKCdelta stimulated cellular differentiation and increased susceptibility of cells to actions of inducers of apoptosis, and they markedly inhibited cellular proliferation and tumor growth in immunodeficient mice. In marked contrast, cPKCbeta and nPKCepsilon increased both in vitro and in vivo growth of cells and inhibited differentiation and apoptosis. Our data present clear evidence for the specific, antagonistic roles of certain cPKC and nPKC isoforms in regulating the above processes in human HaCaT keratinocytes.

Prominent stress response of Purkinje cells in Creutzfeldt-Jakob disease.

To examine the role of stress-related 70-kDa heat shock proteins (Hsp-s) in Creutzfeldt-Jakob disease (CJD), we performed immunocytochemistry to detect Hsp-72 and Hsp-73, together with the abnormal (PrP(Sc)) and the presumed cellular form (PrP(C)) of the prion protein, and TUNEL method to measure cellular vulnerability in different brain regions in CJD and control cases. While Hsp-73 showed uniform distribution in all the examined samples, an increase in the number of Purkinje cells with prominent accumulation of Hsp-72 in the CJD group was observed. These neurons also showed intense PrP(C) staining, but TUNEL-positive nuclei were only detected in the granular (Hsp-72-negative) cell layer. Fewer cells of the inferior olivary nucleus were immunoreactive for Hsp-72 in CJD than in control cases, and regions showing severe spongiform change and gliosis exhibited fewer Hsp-72-immunoreactive neurons. Our results indicate that accumulation of the inducible Hsp-72 in certain cell types may be part of a cytoprotective mechanism, which includes preservation of proteins like PrP(C).

Erythropoietin and erythropoietin receptor expression in human cancer.

Erythropoietin (EPO) stimulates the growth of erythroblasts in the bone marrow (C. Lacombe and P. Mayeux, NEPHROL: DIAL: TRANSPLANT:, 14 (SUPPL: 2): 22-28, 1999). We report basal and hypoxia-stimulated expression of EPO and its receptor, EPOR, in human breast cancer cells, and we demonstrate EPO-stimulated tyrosine phosphorylation and the proliferation of these cells in vitro. In 50 clinical specimens of breast carcinoma, we report high levels of EPO and EPOR associated with malignant cells and tumor vasculature but not with normal breast, benign papilloma, or fibrocystic tissue. Hypoxic tumor regions display the highest levels of EPO and EPOR expression. Enhanced EPO signaling may contribute to the promotion of human cancer by tissue hypoxia.

[Protein kinase C as a potential target for drug development in cancer research]. / A proteinkináz C, mint lehetóség a rákkutatásban specifikus gátlószerek elóállítására.

The central role of protein kinase C (PKC) in cellular signaling makes it an attractive and promising target for drug development. With increasing insight into the regulation by ligands, by phosphorylation, and by macromolecular interactions involved in localization, we will be able to predict the consequences of modifications in second messenger levels resulting from changes in oncogenes, growth factors, or the exposures to xenobiotics. The multiplicity of functionally distinct isoforms of PKC and the depth of modification of their properties in a cell-context specific fashion provide abundant opportunities for generating therapeutic specificity. The emerging elucidation of the complexities of regulation of PKC folding and localization presents the opportunity to pursue novel strategies for intervention, complementing the efforts directed at the design of selective catalytic site inhibitors. Although PKC initially attracted the attention of cancer researchers as a potential therapeutic target, its general role in signal transduction affords a much broader field of opportunity.

Effect of a tyrosine 155 to phenylalanine mutation of protein kinase cdelta on the proliferative and tumorigenic properties of NIH 3T3 fibroblasts.

Tyrosine phosphorylation has emerged as an important mechanism in the regulation of enzyme function. In this paper, we describe a mutant of PKCdelta altered at a single tyrosine residue which has the opposite effect compared with wild-type PKCdelta on the growth characteristics of NIH 3T3 cells. Overexpression of wild-type PKCdelta results in a decreased growth rate and a lower cell density at confluency. On the other hand, overexpression of PKCdelta with a mutation from tyrosine to phenylalanine at position 155 results in a significantly higher rate of growth and a higher density at confluency compared with vector controls. Moreover, these cells are able to grow in soft agar and to form tumors in nude mice. In contrast to kinase negative PKC constructs, this mutant maintains in vitro kinase activity and shows a subcellular localization and a translocation pattern that are similar to those of the wild-type PKCdelta. Whether the altered biological effect is due to the missing phosphorylation on tyrosine or the mutation from tyrosine to phenylalanine per se remains under investigation.

Comparison of the roles of the C1a and C1b domains of protein kinase C alpha in ligand induced translocation in NIH 3T3 cells.

To explore the relative roles of the two C1 domains of protein kinase C alpha (PKC alpha) in the response to phorbol esters and related analogs, we mutated the individual C1 domains, expressed the mutated PKC alpha in NIH 3T3 cells, and then examined the ability of ligands to induce its translocation to the membrane. The C1a and C1b domains play equivalent roles for translocation in response to phorbol 12-myristate 13-acetate, mezerein, and (-)octylindolactam V. These results contrast with those previously reported for PKC delta, suggesting that the domains play different roles in different PKC isoforms.

Regulation of actin cytoskeleton in lymphocytes: PKC-delta disrupts IL-3-induced membrane ruffles downstream of Rac1.

In the murine pre-B lymphoid cell line Baf3, the presence of IL-3 is required for the formation of membrane ruffles that intensely stain for actin and are responsible for the elongated cell phenotype. Withdrawal of IL-3 dissolves ruffled protrusions and converts the cell phenotype to round. Flow cytometric analysis of the cell shape showed that an inactive analog of Rac1 but not inactive RhoA or inactive cdc42 rounds the cells in the presence of IL-3. Constitutively activated Rac1 restores the elongated cell phenotype to IL-3-starved cells. We conclude that the activity of Rac1 is necessary and sufficient for the IL-3-induced assembly of membrane ruffles. Similar to the IL-3 withdrawal, phorbol 12-myristate 13-acetate (PMA) dissolves actin-formed membrane ruffles and rounds the cells in the presence of IL-3. Flow cytometric analysis of the cell shape demonstrated that in the presence of IL-3 the PMA-induced cell rounding cannot be abolished by constitutively active Rac1 but can be imitated by inactive Rac1. These data indicate that PMA disrupts the IL-3 pathway downstream of Rac1. Cells rounded by PMA return to the elongated phenotype concomitantly with PKC depletion. PMA-induced cell rounding can be reversed by the PKC-specific inhibitor GF109203X. Experiments with overexpression in Baf3 of individual PKC isoforms and a dominant negative PKC-delta indicate that activation of PKC-delta but not other PKC isoforms is responsible for disruption of membrane ruffles.

Protein kinase C-epsilon plays a role in neurite outgrowth in response to epidermal growth factor and nerve growth factor in PC12 cells.

In this study, we examined the role of specific protein kinase C (PKC) isoforms in the differentiation of PC12 cells in response to nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells express PKC-alpha, -beta, -gamma, -delta, -epsilon, -mu, and -zeta. For PKC-delta, -epsilon, and -zeta, NGF and EGF exerted differential effects on translocation. Unlike overexpression of PKC-alpha and -delta, overexpression of PKC-epsilon caused enhanced neurite outgrowth in response to NGF. In the PKC-epsilon-overexpressing cells, EGF also dramatically induced neurite outgrowth, arrested cell proliferation, and induced a sustained phosphorylation of mitogen-activated protein kinase (MAPK), in contrast to its mitogenic effects on control cells or cells overexpressing PKC-alpha and -delta. The induction of neurite outgrowth by EGF was inhibited by the MAPK kinase inhibitor PD95098. In cells overexpressing a PKC-epsilon dominant negative mutant, NGF induced reduced neurite outgrowth and a more transient phosphorylation of MAPK than in controls. Our results suggest an important role for PKC-epsilon in neurite outgrowth in PC12 cells, probably via activation of the MAPK pathway.

Protein kinase C delta (PKCdelta) inhibits the expression of glutamine synthetase in glial cells via the PKCdelta regulatory domain and its tyrosine phosphorylation.

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of glial cells. In a recent study we found that overexpression of PKCdelta reduced the expression of the astrocytic marker glutamine synthetase (GS). In this study we explored the mechanisms involved in the inhibitory effect of PKCdelta on the expression of glutamine synthetase. Using PKC chimeras we first examined the role of the catalytic and regulatory domains of PKCdelta on the expression of glutamine synthetase. We found that cells stably transfected with chimeras between the regulatory domain of PKCdelta and the catalytic domains of PKCalpha or epsilon inhibited the expression of GS, similar to the inhibition exerted by overexpression of PKCdelta itself. In contrast, no significant effects were observed in cells transfected with the reciprocal PKC chimeras between the regulatory domains of PKCalpha or epsilon and the catalytic domain of PKCdelta. PKCdelta has been shown to undergo tyrosine phosphorylation in response to various activators. Tyrosine phosphorylation of PKCdelta in response to phorbol 12-myristate 13-acetate and platelet-derived growth factor occurred only in chimeras which contained the PKCdelta regulatory domain. Cells transfected with a PKCdelta mutant (PKCdelta5), in which the five putative tyrosine phosphorylation sites were mutated to phenylalanine, showed markedly diminished tyrosine phosphorylation in response to phorbol 12-myristate 13-acetate and platelet-derived growth factor and normal levels of GS. Our results indicate that the regulatory domain of PKCdelta mediates the inhibitory effect of this isoform on the expression of GS. Phosphorylation of PKCdelta on tyrosine residues in the regulatory domain is implicated in this inhibitory effect.

Specific vanilloid responses in C6 rat glioma cells.

Capsaicin and its ultrapotent analog resiniferatoxin (RTX) act through specific vanilloid receptors on sensory neurons. Here, we describe specific vanilloid responses in rat C6 glioma cells. Capsaicin and RTX stimulated 45Ca uptake in a similar fashion to that found for cultured rat dorsal root ganglion neurons (DRGs); this response was antagonized by the antagonists capsazepine and ruthenium red. As in DRGs, pretreatment of C6 cells with capsaicin or RTX produced desensitization to subsequent stimulation of 45Ca uptake. The potency for desensitization by RTX in the C6 cells corresponded to that for 45Ca uptake, whereas in DRGs it occurred at significantly lower concentrations corresponding to that for the high affinity [3H]RTX binding site. Consistent with this difference, in C6 cells we were unable to detect [3H]RTX binding. These characteristics suggest the presence of C-type but not R-type vanilloid receptors on C6 cells. After 2 day treatment, capsaicin but not RTX inhibited the proliferation and altered the differentiation of the cells and produced apoptosis. In the long term experiments, capsazepine, instead of antagonizing the effect of capsaicin, acted as an agonist. Moreover, capsazepine displayed these effects with higher potency than that of capsaicin. The different potencies and structure activity relations suggest a distinct mechanism for these long-term vanilloid effects. Our finding that C6 cells can respond directly to capsaicin necessitates a reevaluation of the in vivo pathway of response to vanilloids, and highlights the importance of the neuron-glial network.

Differential role of specific PKC isoforms in the proliferation of glial cells and the expression of the astrocytic markers GFAP and glutamine synthetase.

In this study, we explored the role of specific protein kinase C (PKC) isoforms in glial cell proliferation and on the expression of the astrocytic markers GFAP and glutamine synthetase using C6 cells as a model. Analysis of the expression of the various PKC isoforms in control and differentiated C6 cells revealed differences in the expression of specific PKC isoforms. Undifferentiated C6 cells, which express low levels of GFAP and glutamine synthetase (GS), have high levels of PKCalpha and delta, whereas differentiated C6 cells, which express higher levels of both GFAP and GS have lower levels of PKCalpha and delta and higher levels of PKCgamma, theta and eta. Using C6 cells overexpressing specific PKC isoforms, we examined the role of these isoforms on the proliferation and differentiation of C6 cells. Cells overexpressing PKCalpha displayed a reduced level of GFAP, whereas GS expression was not affected. On the other hand, cells overexpressing PKCdelta showed reduced GS expression but little effect on GFAP. Finally, cells expressing PKCgamma displayed a marked increase in the levels of both GFAP and GS. The proliferation of C6 cells was increased in cells overexpressing PKCalpha and epsilon and decreased in cells overexpressing PKCgamma, delta and eta. The results of this study suggest that glial cell proliferation and astrocytic differentiation can be regulated by specific PKC isoforms that selectively affect cell proliferation and the expression of the two astrocytic markers GFAP and GS.

Differential selectivity of ligands for the C1a and C1b phorbol ester binding domains of protein kinase Cdelta: possible correlation with tumor-promoting activity.

Protein kinase C (PKC) represents the major, high-affinity receptor for the phorbol esters as well as for a series of structurally diverse natural products. The phorbol esters function by binding to the tandem C1a and C1b domains in PKC, leading to enzyme activation. Although the typical phorbol esters represent the paradigm for tumor promoters in mouse skin, it is now clear that different high affinity ligands for PKC have distinct biological effects. Thus, the daphnane analogue mezerein is a second-stage promoter, the macrolide bryostatin 1 is a partial antagonist, and certain 12-deoxyphorbol 13-monoesters also function as partial antagonists but with a different pattern of activity. The biochemical basis for these differences is an area of active investigation. In this report, we have examined the relative interaction of ligands differing in structure and pattern of biological response with the C1a and C1b domains of PKCdelta. We mutated either or both of the C1 domains of PKCdelta, expressed the constructs in NIH 3T3 cells, and monitored the interaction of the ligands by their ability to induce translocation of the mutated PKCdelta from the cytosol to the particulate fraction. We found that different ligands showed different dependence on the C1a and C1b domains for translocation. Whereas phorbol 12-myristate 13-acetate and the indole alkaloids indolactam and octylindolactam were selectively dependent on the C1b domain, selectivity was not observed for mezerein, for the 12-deoxyphorbol 13-monoesters prostratin or 12-deoxyphorbol 13-phenylacetate, or for the macrocyclic lactone bryostatin 1. Provocatively, the pattern of response corresponds with the activity of the compounds as complete tumor promoters.

Characterization of functional vanilloid receptors expressed by mast cells.

Capsaicin and its ultrapotent analog resiniferatoxin (RTX) act through specific vanilloid receptors on sensory neurons. The C-type receptor is coupled to 45Ca uptake, whereas the R-type is detectable by [3H]RTX binding. We describe here specific vanilloid responses in murine mast cells (MCs). In the MC lines and in bone marrow-derived mast cells, capsaicin and RTX induced 45Ca uptake similarly to that observed for cultured rat dorsal root ganglion neurons (DRGs). This response was antagonized by the antagonists capsazepine and ruthenium red. As in DRGs, pretreatment of MCs with capsaicin or RTX induced desensitization to subsequent stimulation of 45Ca uptake. The potency for desensitization by RTX in the MCs corresponded to that for 45Ca uptake, whereas in DRGs it occurred at significantly lower concentrations corresponding to that for the high-affinity [3H]RTX binding site. Consistent with this difference, in MCs we were unable to detect [3H]RTX binding. Vanilloids were noncytotoxic to the MCs, in contrast to the DRGs. Although vanilloids did not cause degranulation in MCs, in the P815 clone capsaicin evoked selective interleukin-4 release. We conclude that certain MCs possess vanilloid receptors, but only the C-type that functions as a channel. Our finding that MCs can respond directly to capsaicin necessitates a reevaluation of the in vivo pathway of inflammation in response to vanilloids.

The catalytic domain of PKC-epsilon, in reciprocal PKC-delta and -epsilon chimeras, is responsible for conferring tumorgenicity to NIH3T3 cells, whereas both regulatory and catalytic domains of PKC-epsilon contribute to in vitro transformation.

Protein kinase C-epsilon (PKC-epsilon) has been shown to increase growth and cause malignant transformation when overexpressed in NIH3T3 cells, whereas PKC-delta reduced fibroblast growth. Two reciprocal chimeric proteins (PKC-epsilondelta and PKC-deltaepsilon were constructed by exchanging the regulatory and catalytic domains of PKC-delta and -epsilon and were stably overexpressed in NIH3T3 cells. Fibroblasts that overexpressed either chimera showed maximum cell density and morphology that were intermediate between cells overexpressing PKC-delta and those that overexpressed PKC-epsilon. Moreover, all lines that expressed chimeras were capable of anchorage-independent growth in the presence of TPA, which indicated that both the regulatory and catalytic domains of PKC-epsilon could independently induce NIH3T3 transformation, although the combination of both domains, as found in PKC-epsilon, was the most active form. In contrast, the translocation pattern and ability to induce tumors in nude mice was attributable to the catalytic domains exclusively. In particular, cells that expressed PKC-deltaepsilon retained PKC-epsilon's full potency of tumorgenicity when injected into nude mice. In sum, our findings not only reinforce the concept that only certain PKC isozymes contribute to carcinogenesis but also show that different domains of PKCs mediate the physiologically distinguishable events of transformation and tumorgenesis.

Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH 3T3 cells.

Protein kinase C (PKC) isozymes exhibit important differences in terms of their regulation and biological functions. Not only may some PKC isoforms be active and others not for a given response, but the actions of different isoforms may even be antagonistic. In NIH 3T3 cells, for example, PKCdelta arrests cell growth whereas PKCepsilon stimulates it. To probe the contribution of the regulatory and the catalytic domains of PKC isozymes to isozyme-specific responses, we prepared chimeras between the regulatory and the catalytic domains of PKCalpha, -delta, and -epsilon. These chimeras, which preserve the overall structure of the native PKC enzymes, were stably expressed in mouse fibroblasts. A major objective was to characterize the growth properties of the cells that overexpress the various PKC constructs. Our data demonstrate that both the regulatory and the catalytic domains play roles in cell proliferation. The regulatory domain of PKCepsilon enhanced cell growth in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and, in the presence of PMA, all chimeras with the PKCepsilon regulatory domain also gave rise to colonies in soft agar; the role of the catalytic domain of PKCepsilon was evident in the PMA-treated cells that overexpressed the PKC chimera containing the delta regulatory and the epsilon catalytic domains (PKCdelta/epsilon). The important contribution of the PKCepsilon catalytic domain to the growth of PKCdelta/epsilon-expressing cells was also evident in terms of a significantly increased saturation density in the presence of PMA, their formation of foci upon PMA treatment, and the induction of anchorage-independent growth. Aside from the growth-promoting effect of PKCepsilon, we have shown that most chimeras with PKCalpha and -delta regulatory domains inhibit cell growth. These results underscore the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC.

Functional effects of overexpression of protein kinase C-alpha, -beta, -delta, -epsilon, and -eta in the mast cell line RBL-2H3.

The rat basophilic leukemic (RBL-2H3) cell line was stably transfected with the endogenously expressed Ca2+-dependent protein kinase C-alpha (PKC-alpha) and -betaI and the Ca2+-independent delta and epsilon isoforms to study their functional roles. In addition, the Ca2+-independent PKC-eta was expressed. All transfected PKC isoforms translocated to the membrane-containing fraction in response to aggregation of the IgE-sensitized high affinity receptor for IgE (Fc epsilonRI) with the Ag dinitrophenyl(25)-BSA. All PKC transfectants, except PKC-eta, showed increased proliferative responses, and aggregation of Fc epsilonRI further enhanced the rate of proliferation. The PKC transfectants also showed increased phosphoinositide hydrolysis in response to Ag aggregation of receptors. No marked differences in the Ca2+ responses of the transfectants to Ag or thapsigargin were observed. Overexpression of PKC-alpha or -epsilon specifically inhibited receptor-dependent cytosolic phospholipase A2 (cPLA2) activity, whereas this activity was enhanced in the PKC-betaI transfectant. Analysis of the secretory response revealed that overexpression of PKC-betaI and -eta significantly enhanced secretion. A broad spectrum of cytokine mRNAs was detected in all transfectants, and overexpression of PKC-betaI significantly enhanced the receptor-dependent production of IL-2 and IL-6 mRNA. These studies identify PKC-alpha and -epsilon as negative regulators of cPLA2 activity and demonstrate the importance of PKC-beta as a positive modulator of secretion, cPLA2 activity, and cytokine production in this mast cell line.