Benzyl isothiocyanate inhibits IL-13 expression in human basophilic KU812 cells.

An allergen-stimulating cytokine, interleukin-13 (IL-13), plays a significant role in allergic inflammation. Benzyl isothiocyanate (BITC), derived from several cruciferous vegetables, significantly suppressed the IL-13 expression in the calcium ionophore-stimulated human basophilic KU812 cells. Down-regulation of phosphorylated mitogen-activated protein kinases as well as nuclear transcriptional factors might be involved in the underlying mechanism.

Butrin, isobutrin, and butein from medicinal plant Butea monosperma selectively inhibit nuclear factor-kappaB in activated human mast cells: suppression of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8.

Activation of mast cells in rheumatoid synovial tissue has often been associated with tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 production and disease pathogenesis by adjacent cell types. Butea monosperma (BM) is a well known medicinal plant in India and the tropics. The aim of this study was to examine whether a standardized extract of BM flower (BME) could inhibit inflammatory reactions in human mast cells (HMC) using activated HMC-1 cells as a model. Four previously characterized polyphenols--butrin, isobutrin, isocoreopsin, and butein--were isolated from BME by preparative thin layer chromatography, and their purity and molecular weights were determined by liquid chromatography/mass spectrometry analysis. Our results showed that butrin, isobutrin, and butein significantly reduced the phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced inflammatory gene expression and production of TNF-alpha, IL-6, and IL-8 in HMC-1 cells by inhibiting the activation of NF-kappaB. In addition, isobutrin was most potent in suppressing the NF-kappaB p65 activation by inhibiting IkappaBalpha degradation, whereas butrin and butein were relatively less effective. In vitro kinase activity assay revealed that isobutrin was a potent inhibitor of IkappaB kinase complex activity. This is the first report identifying the molecular basis of the reported anti-inflammatory effects of BME and its constituents butrin, isobutrin, and butein. The novel pharmacological actions of these polyphenolic compounds indicate potential therapeutic value for the treatment of inflammatory and other diseases in which activated mast cells play a role.

Ellagic acid inhibits lipopolysaccharide-induced expression of enzymes involved in the synthesis of prostaglandin E2 in human monocytes.

Ellagic acid, a natural polyphenol found in certain fruits, nuts and vegetables, has in recent years been the subject of intense research within the fields of cancer and inflammation. Pain, fever and swelling, all typical symptoms of inflammation, are ascribed to elevated levels of PGE2. In the present study, we have investigated the effects of ellagic acid on PGE2 release and on prostaglandin-synthesising enzymes in human monocytes. Ellagic acid was found to inhibit Ca ionophore A23187-, phorbol myristate acetate- and opsonised zymosan-induced release of PGE2 from monocytes pre-treated with the inflammatory agent lipopolysaccharide. Ellagic acid suppressed the lipopolysaccharide-induced increase in protein expression of cyclo-oxygenase-2 (COX-2), microsomal PGE synthase-1 (mPGEs-1) and cytosolic phospholipase A2alpha (cPLA2alpha), while it had no effect on the constitutively expressed COX-1 protein. Ellagic acid had no apparent inhibitory effect on these enzymes when the activities were determined in cell-free assays. We conclude that the inhibitory effect of ellagic acid on PGE2 release from monocytes is due to a suppressed expression of COX-2, mPGEs-1 and cPLA2alpha, rather than a direct effect on the activities of these enzymes.

Blockade of cytosolic phospholipase A(2) and 5-lipoxygenase activation in neutrophils by a natural isoflavanquinone abruquinone A.

Abruquinone A, a natural isoflavanquinone, suppressed A23187- and formyl-Met-Leu-Phe (fMLP)-induced production of thromboxane B(2) and leukotriene B(4) from rat neutrophils. This compound failed to inhibit the enzymatic activity of ram seminal vesicles cyclooxygenase (COX) and human recombinant 5-lipoxygenase (5-LO) in cell-free systems. Abruquinone A diminished the arachidonic acid release from [(3)H]arachidonic acid-loaded neutrophils stimulated with either fMLP or A23187, whereas it had no inhibitory effect on the cytosolic phospholipase A(2) (cPLA(2)) activity of neutrophil cytosolic fraction. Based on the Western blot analysis, the nuclear membrane recruitment of cPLA(2) and 5-LO was inhibited by abruquinone A in A23187- as well as in fMLP-stimulated cells. Moreover, the phosphorylation of both cPLA(2) and extracellular signal regulated kinases (ERKs) induced by fMLP and A23187 was attenuated by abruquinone A in a parallel concentration-dependent manner. Abruquinone A attenuated both fMLP- and ionomycin-mediated [Ca(2+)](i) elevation in a concentration range that inhibited the recruitment of cPLA(2) to nuclear membrane. These results indicate that the blockade of leukotriene B(4) production by abruquinone A implicates the attenuation of 5-LO membrane translocation. Inhibition of thromboxane B(2) production by abruquinone A is due to the attenuation of cPLA(2) membrane recruitment and/or cPLA(2) phosphorylation through the blockade of [Ca(2+)](i) elevation and ERK activation, respectively.

Cytoprotective roles of senescence marker protein 30 against intracellular calcium elevation and oxidative stress.

Senescence marker protein 30 (SMP30) is identified as an important aging marker molecule and known to play multifunctional roles as an intracellular calcium regulatory protein in the signaling process. To elucidate the functional significance of SMP30, we established the stably transfected P19 cell line with SMP30 expression vector. Overexpression of SMP30 slightly suppressed the proliferation of P19 cells. However, SMP30 overexpression was cytoprotective against calcium-mediated stress such as calcium ionophore (A23187), and thapsigargin. We found that SMP30 overexpression reduced the elevated intracellular calcium levels induced by A23187, but not by thapsigargin. In addition, SMP30 transfected P19 cells were more protective to tert-butylhydroperoxide induced cytotoxicity, indicating the antioxidative properties of SMP30. Taken together, our results suggest that external calcium regulation and antioxidant properties are involved in the cytoprotective mechanism of SMP30.

Effect of calcium ionophore A23187 on prostaglandin synthase type 2 and 15-hydroxy-prostaglandin dehydrogenase expression in human chorion trophoblast cells.

OBJECTIVE: Prostaglandins induce parturition in humans. Prostaglandin output is regulated by the synthetic and metabolic enzymes, prostaglandin synthase type 2 (PTGS2) and 15-hydroxyprostaglandin dehydrogenase (PGDH). The role of calcium in regulating PTGS2 and PGDH expression was investigated in chorion trophoblasts. STUDY DESIGN: Cells were treated with calcium ionophore A23187 in the presence or absence of calcium chelators; changes in messenger ribonucleic acid expression were measured with real-time polymerase chain reaction and analyzed with analysis of variance. Protein expression was evaluated with Western blot and dual immunofluorescence. RESULTS: A23187 stimulated PTGS2 and suppressed PGDH expression. Effects of A23187 were reversed by calcium chelators. PTGS2 had perinuclear and cytosolic distribution, whereas PGDH was cytosolic. Some cells expressed both enzymes, some neither enzyme, and some either PTGS2 or PGDH. CONCLUSION: Chorion cells showed heterogeneity in the expression of PTGS2 and PGDH. Calcium influx regulates PTGS2 and PGDH expression, thereby promoting coordinated increased prostaglandin output in circumstances such as term and preterm labor.

Mechanism of A23187-induced apoptosis in HL-60 cells: dependency on mitochondrial permeability transition but not on NADPH oxidase.

Calcium ions (Ca(2+)) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca(2+) in A23187-treated HL-60 cells was associated with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (MPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca(2+) induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of MPT pores that leads to apoptotic cell death.

Divergent regulation of Pyk2/CAKbeta phosphorylation by Ca2+ and cAMP in the hippocampus.

Proline-rich tyrosine kinase 2 (Pyk2) is activated in neurones following NMDA receptor stimulation via PKC. Pyk2 is involved in hippocampal LTP and acts to potentiate NMDA receptor function. Elevations of intracellular Ca2+ and cAMP levels are key NMDA receptor-dependent triggering events leading to induction of hippocampal LTP. In this study, we compared the ability of A23187 (Ca2+ ionophore) or forskolin (adenylate cyclase activator) to modulate the phosphorylation of Pyk2 in rat hippocampal slices. Using an immunoprecipitation assay, phosphorylated Pyk2 levels were increased following treatment with A23187, levels peaking at around 10 min. Staurosporine, at concentrations inhibiting conventional and novel isoforms of PKC, and chelerythrine, at concentrations inhibiting the atypical PKC isoform PKMxi, were compared for their ability to attenuate the effect of A23187. Exposure of acute hippocampal slices to either chelerythrine or staurosporine completely blocked enhanced phosphorylation of Pyk2 by A23187, suggesting a possible involvement of PKMxi and typical PKCs in Pyk2 activation by Ca2+. In contrast, application of forskolin reduced phosphorylated Pyk2 below basal levels, suggesting that cAMP inhibits Pyk2. These results implicate Ca2+ and multiple forms of PKC in the activation of Pyk2 downstream of NMDA receptors and suggest that cAMP-dependent processes exert a suppressive action on Pyk2.

The phenoxazine derivative Phx-1 suppresses IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells.

Antigen-induced aggregation of the high affinity IgE receptor (FcepsilonRI) on mast cells induces degranulation to release chemical mediators, leading to acute allergic inflammation. We have demonstrated that the treatment of rat mast cells, RBL-2H3, with a phenoxazine derivative Phx-1 (2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one) suppresses the antigen-induced degranulation. Biochemical analysis reveals that the complementary signaling pathway through Gab2 and Akt is inhibited by this compound in mast cells. These findings suggest that phenoxazine derivatives may have a therapeutic potential for allergic diseases by inhibiting mast cell degranulation.

Multiple signalling pathways mediate fungal elicitor-induced beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures.

The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of beta-thujaplicin. The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin. A23187 also enhanced the elicitor-induced production of beta-thujaplicin. EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin. These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity. These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.

The effect of conjugated linoleic acid on arachidonic acid metabolism and eicosanoid production in human saphenous vein endothelial cells.

The effects of a conjugated linoleic acid (CLA) mixture of single isomers (50:50, w/w, cis9,trans11:trans10,cis12) and the individual isomers on (a) the production of resting and calcium ionophore stimulated (14)C-eicosanoids and (b) the incorporation of (14)C-arachidonic acid (AA) into membrane phospholipids of human saphenous vein endothelial cells were investigated. The CLA mixture and the individual isomers were found to inhibit resting production of (14)C-prostaglandin F(2a) by 50, 43 and 40%, respectively. A dose dependent inhibition of stimulated (14)C-prostaglandins was observed with the CLA mixture (IC(50) 100 microM). The cis9,trans11 and trans10,cis12 (50 microM) isomers individually inhibited the overall production of stimulated (14)C-prostaglandins (between 35 and 55% and 23 and 42%, respectively). When tested at a high concentration (100 microM), cis9,trans11 was found to inhibit eicosanoid production in contrast to trans10,cis12 that caused stimulation. The overall degree of (14)C-AA incorporation into membrane phospholipids of the CLA (mixture and individual isomers) treated cells was found to be lower than that of control cells and the cis9,trans11 isomer was found to increase the incorporation of (14)C-AA into phosphatidylcholine. Docosahexaenoic acid, eicosapentaenoic acid and linoleic acid did not alter the overall degree of incorporation of (14)C-AA. The results of this study suggest that both isomers inhibit eicosanoid production, and although trans10,cis12 exhibits pro-inflammatory activity at high concentrations, the CLA mixture maintains its beneficial anti-inflammatory action that contributes to its anti-carcinogenic and anti-atherogenic properties.

Effect of H1-antagonist Dithiaden on human PMN-leukocyte aggregation and chemiluminescence is stimulus-dependent.

OBJECTIVE AND DESIGN: Contradictory data published on histamine-PMN leukocyte interactions stimulated us to study to the role of histamine and H1-antagonist Dithiaden in generation of reactive oxygen species (ROS) and aggregation of human neutrophils. METHODS AND MATERIALS: Whole blood or isolated PMN-leukocytes were exposed in a dose-dependent way to histamine or H1-antagonist Dithiaden and subsequently stimulated. Whole blood was stimulated with opsonised zymosan (OZ). Isolated cells were stimulated with membrane stimuli (OZ, N-formyl-methionyl-leucyl-phenylalanine--fMLP), or membrane bypassing stimuli (Ca2+-ionophore A23187, phorbol-myristate-acetate--PMA). The luminol-enhanced chemiluminescence (CL) was measured separately (whole blood) in a luminometer or simultaneously with neutrophil aggregation in a whole blood lumiaggregometer. RESULTS: Depending on the concentration used, Dithiaden" was 1.5- to 25.0-times more effective in inhibiting activated CL of whole blood than histamine. In isolated neutrophils both histamine and Dithiaden inhibited OZ- and A23187-stimulated CL dose-dependently, with potentiation observed after stimulation with PMA and fMLP. Histamine did not alter aggregation with any of the stimuli tested. Dithiaden inhibited A23187-, OZ- and PMA-stimulated PMN-leukocytes but potentiated fMLP-induced aggregation of isolated neutrophils. Simultaneous application of Dithiaden and histamine abolished the effect of Dithiaden on fMLP-stimulated CL. CONCLUSIONS: Dithiaden, depending on the stimuli applied, inhibited human neutrophils, both isolated or in whole blood, more markedly than histamine. The inhibition of aggregation and CL was dose- and stimulus-dependent. Histamine administered simultaneously abolished the effect of Dithiaden on fMLP-stimulated PMN-leukocytes. It seems likely that the interaction of Dithiaden with neutrophils operated both at an extra- and intracellular level.

Wortmannin inhibits platelet aggregation produced by interaction of gamma-aminobutyric acid and the calcium tonophore, A23187.

Platelet aggregation by gamma-aminobutyric acid (GABA) agonists combined with a calcium ionophore was studied. GABA, baclofen and mucimol markedly amplified aggregatory responses to a subthreshold concentration of the ionophore, A23187. This effect was inhibited by wortmannin, a blocker of phosphoinositide 3-kinase. However, several antagonists of GABA receptors had no effect on the response, and benzodiazepines inhibited aggregation. These results suggest that the GABA effect is not mediated by traditional neuronal GABA receptors. We propose that wortmannin inhibits aggregation at a nexus downstream from membrane mechanisms triggered by the GABA-A23187 interaction.

Estrogen regulates cytokine release in human mast cells.

Estrogens are important for bone homeostasis and are classified as anti-resorptive agents. In ovariectomized rats, mast cell changes occurred during the activation of resorption. In addition, quantitative changes occurred in mast cell population residing near the site undergoing resorption. Considering these studies, mast cells may play a role in osteoporosis. Therefore, it is of paramount importance to study mast cell cytokine production also in the presence or absence of estrogen. When cultured in the absence of estrogen, human mast cells treated with PMA or A23187 demonstrated significantly greater release of TNF-alpha and IL-6 than cells grown under estrogen-depleted condition. Our results show that treatment of mast cells with estrogen prevented PMA or A23187-stimulated TNF-alpha or IL-6 release. These data provide evidence for a potent inhibition of cytokines by estrogen in human mast cells. This study may help to explain the association between mast cells and osteoporosis.

Regulatory mechanism of eosinophil peroxidase release from guinea pig eosinophils.

The regulatory mechanism of degranulation of guinea pig peritoneal eosinophils was studied by determination of eosinophil peroxidase (EPO) release. Beta-agonists, such as isoproterenol, salbutamol and fenoterol, effectively inhibited A23187-induced EPO release from guinea pig eosinophils. The inhibitory effects of beta-agonists were attenuated by pretreatment with either propranolol, a non-selective beta-antagonist, or ICI 118,551, a selective beta2-antagonist. Both theophylline and dibutyryl-cAMP (db-cAMP) also significantly inhibited A23187-induced EPO release. The inhibition of EPO release induced by db-cAMP was attenuated by pretreatment with KT5720, a protein kinase A inhibitor. In addition, calphostin C as well as cytochalasin D effectively inhibited A23187-induced EPO release. From the results of the present study, it was concluded that an increase in intracellular Ca2+ concentration may lead to exocytosis of eosinophil granules through activation of protein kinase C and microfilaments. Beta-agonists and theophylline were effective in inhibiting degranulation of eosinophils by increasing intracellular cAMP level coupled with the activation of protein kinase A.

Deencryption of cellular tissue factor is independent of its cytoplasmic domain.

Tissue factor (TF) is a transmembrane molecule that, when exposed to plasma, is the key initiator of coagulation. Cellular TF activity is normally "encrypted", but treating cells with calcium ionophore (i.e. , ionomycin or A23187) increases ("deencrypts") TF activity without increasing TF mRNA or antigen expression. Deencryption results from both plasma membrane phosphatidylserine (PS)-dependent and -independent mechanisms; however, the nature of the PS-independent component is unclear. Since deencryption has been suggested to result from release of TF dimers on the cell surface, and since TF's cytoplasmic domain binds to actin-binding protein 280 and interacts with the cytoskeleton, we hypothesized that interactions with the cytoskeleton, through the cytoplasmic domain, play a role in mediating encryption/deencryption. We examined TF deencryption and the role of the cytoplasmic domain in the PS-independent component using baby hamster kidney (BHK) cells expressing full length TF (BHK-TF) or TF lacking its cytoplasmic domain (BHK-descyt) (Sorensen et al. (1999) J. Biol. Chem. 274, 21349). Both BHK-TF and BHK-descyt cells exhibited a dose-dependent, 1.5- to 10-fold increase in TF activity upon treatment with calcium ionophore, and this increase in activity was only partially blocked by annexin V. These results indicate that deencryption is not restricted to cells which naturally express TF and that the PS-independent component of deencryption is intact on cells transfected with either full length or truncated TF. Our results clearly indicate that deencryption is not dependent on an intact cytoplasmic domain in transfected BHK cells.

T cell activation up-regulates the expression of the focal adhesion kinase Pyk2: opposing roles for the activation of protein kinase C and the increase in intracellular Ca2+.

T cell activation initiates signals that control gene expression of molecules important for T cell function. The focal adhesion kinase Pyk2 has been implicated in T cell signaling. To further analyze the involvement of Pyk2 in T cell processes, we examined the effect of T cell stimulation on the expression of Pyk2. We found that TCR ligation or PMA increased Pyk2 expression in Jurkat T cells and in normal T cells. In contrast, TCR ligation and PMA failed to induce any detectable increase in the expression of the other member of the focal adhesion kinase family, Fak, in Jurkat T cells and induced only a weak increase in Fak expression in normal T cells. The serine/threonine kinases, protein kinase C and mitogen-activated protein/extracellular signal-related kinase kinase (MEK), regulated Pyk2 expression, as inhibitors of these kinases blocked stimulus-induced Pyk2 expression. Cyclosporin A, FK506, and KN-62 did not block Pyk2 expression; thus, calcineurin and Ca2+/calmodulin-activated kinases are not critical for augmenting Pyk2 expression. TCR ligation increased Pyk2 mRNA, and the transcriptional inhibitor actinomycin D blocked Pyk2 expression. Strikingly, Ca2+ ionophores, at concentrations that in combination with other stimuli induced IL-2 expression, blocked TCR- and PMA-induced up-regulation of Pyk2 expression. Thus, the increase in Ca2+ has opposing effects on IL-2 and Pyk2 expression. Cyclosporin A and FK506, but not KN-62, blocked Ca2+ ionophore-mediated inhibition of Pyk2 expression, implicating calcineurin in down-regulating Pyk2 expression. These results show that TCR-triggered intracellular signals increase Pyk2 expression and shed light on the molecular mechanisms that regulate Pyk2 expression in T cells.

Cytosolic phospholipase A2-mediated regulation of phospholipase D2 in leukocyte cell lines.

Phospholipase D (PLD) has been implicated in a variety of cellular processes, including inflammation, secretion, and respiratory burst. Two distinct PLD isoforms, designated PLD1 and PLD2, have been cloned; however, the regulatory mechanism for each PLD isoform is not clear. In our present study we investigated how PLD2 activity is regulated in mouse lymphocytic leukemia L1210 cells, which mainly contain PLD2, and in PLD2 -transfected COS-7 cells. Intriguingly, A23187, a calcium ionophore that induces calcium influx, potently stimulates PLD activity in these two cell lines, suggesting that Ca2+ might be implicated in the regulation of the PLD2 activity. In addition to the A23187-induced PLD2 activation, A23187 also increases PLA2-mediated arachidonic acid release, and the A23187-stimulated PLD2 and PLA2 activities could be blocked by pretreatment of the cells with cytosolic calcium-dependent PLA2 (cPLA2) inhibitors, such as arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate in these two cell lines. Moreover, the A23187-induced PLD2 and PLA2 activities could be inhibited by cotransfection with antisense cPLA2 oligonucleotide. These results suggest a role for cPLA2 in the regulation of PLD2 activity in vivo. The inhibitory effect of arachidonyl trifluoromethyl ketone on the A23187-induced PLD2 activity could be recovered by addition of exogenous lysophosphatidylcholine. This study is the first to demonstrate that PLD2 activity is up-regulated by Ca2+ influx and that cPLA2 may play a key role in the Ca2+-dependent regulation of PLD2 through generation of lysophosphatidylcholine.

Secretion of metalloendopeptidase 24.15 (EC 3.4.24.15).

The metalloendopeptidase EP24.15 (EC3.4.24.15) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly. Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment. We utilized a model system of neuroendocrine secretion, the AtT20 cell. According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested that the mechanism of EP24.15 secretion is not solely via classical secretory pathways. Additionally, drugs which disrupt the classical secretory pathway, such as Brefeldin A and nocodazole, blocked A23187-stimulated EP24.15 release yet had no effect on basal EP24.15 release, suggesting differences in the basal and stimulated pathways of secretion for EP24.15. In summary, EP24.15 appears to be secreted from AtT20 pituitary cells into the extracellular milieu, where the enzyme can participate in the physiologic metabolism of neuropeptides.

Effects of TGF-beta1 on prolactin synthesis and secretion: an in-vitro study.

The hypothalamus exerts a predominantly inhibitory influence on prolactin secretion through dopamine. In addition, the expression of anterior pituitary hormone-gene products are regulated by intrapituitary growth factors. In particular, TGF-beta1 produced in the pituitary regulates lactotroph cell proliferation and prolactin gene-expression. This study characterized the regulation of in-vitro prolactin synthesis and secretion by TGF-beta1 using rat anterior pituitary cells in monolayer culture. Furthermore, we studied the interaction of TGF-beta1 with other signals involved in the neuroregulation of prolactin secretion, such as dopamine and TRH, as well as the importance of different signal transduction pathways in this response. TGF-beta1 inhibited prolactin secretion in a time- and concentration-dependent manner, with half-maximal inhibition occurring at the range of 15-30 pM. The inhibitory effect was observed after 4 h, being maximal after 4 days of exposure of the cells to the peptide. This inhibitory effect was mimicked by TGF-beta2 but not by inhibin, and was not influenced by oestrogens, being similar in male, normal female or oestradiol-treated rats. Prolonged pretreatment of the cells with TGF-beta1(4 days) did not modify GH or TSH secretion nor dopamine-induced inhibition of prolactin secretion, and blunted prolactin responses to TRH, Forskolin, But2-cAMP and to the calcium ionophore A23187. The effect observed after long-term treatment (24 h to 4 days) is essentially caused by a decrease in prolactin synthesis, since TGF-beta1 inhibited prolactin mRNA levels and de novo prolactin protein synthesis. However, in the short term (up to 12 h) TGF-beta1 inhibition of prolactin secretion was associated with an increase in intracellular prolactin content, dissecting a dual mechanism of action of TGF-beta1. The short-term TGF-beta1 effect did not modify Erk-2 phosphorylation, basal or TRH-induced increase in intracellular calcium concentration, but blunted basal and forskolin stimulated cAMP levels. But2-cAMP replacement did not revert the inhibition of prolactin secretion. However, pertussis toxin was able to recover a large percentage of TGF-beta1-induced inhibition of prolactin secretion. This study indicates that TGF-beta1 plays a crucial role as a modulator of lactotroph function, inhibiting prolactin biosynthesis after long-term treatment, as well as, after short-term exposure prolactin secretion at the level of the secretory process, through a mechanism pertussis toxin sensitive but independent of Erk-2 phosphorylation, calcium concentrations or intracellular cAMP.