Biomedical subjects

B Haye

Publications and source records attributed to B Haye.

At least 19 recordsLinked to original sources

PubMed1992

Autocrine biological effects of glycosyl inositol phosphate produced by reconstituted pig thyroid follicles: role of pertussis toxin sensitive G proteins.

We examine the autocrine activity of glycosyl inositol phosphate (InP-gly) on thyroid metabolism. The cAMP accumulation promoted by thyrotropin (TSH) or forskolin was modulated by InP-gly, stimulated by the lowest tested concentration (10(-8) M) and progressively inhibited by higher concentrations. Iodide uptake and iodine organification were decreased in a concentration-dependent manner by InP-gly alone, or in the presence of TSH. The IAP component of pertussis toxin blocked the inhibitory action of InP-gly on cAMP accumulation by reconstituted thyroid follicles (RTF), suggesting the participation of Gi protein. But the same treatment with IAP was without effect on iodine metabolism, suggesting that there is a second target for InP-gly, more distal than Gi protein, or coupled to another G protein which is insensitive to the toxin.

Animals↗

PubMed1991

The effect of glycosyl inositol-phosphate on cAMP production in isolated rat fat-cells is transduced by a pertussis toxin sensitive G-protein.

In fat-cells, insulin increases synergistically the inhibitory effects of adenosine and prostaglandin E2 on adenylyl cyclase activity. When the endogenous production of these feedback inhibitors is suppressed, insulin is no more active in conditions where glycosyl inositol-phosphate which is a putative mediator of its action, is always efficient. Moreover, glycosyl inositol-phosphate signal is transduced by a G protein sensitive to IAP intoxication.

Adenosine↗

PubMed1991

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1991

Alteration of the functional activity of Gs protein in thyrotropin-desensitized pig thyroid cells.

Changes in the sensitivity of adenylyl cyclase observed in pig thyroid cells cultured 2 days in the presence of thyroid-stimulating hormone (TSH) or forskolin were assessed by examining the properties of Gs protein. Chronic treatment of thyroid cells with various concentrations of TSH (0.01-1 mU/ml) or forskolin (0.1-10 microM) increased the response of adenylyl cyclase to a further stimulation by forskolin or NaF + AlCl3 ([AlF4]-). In contrast, the enzyme activation promoted by guanosine 5'-(beta,gamma-imido) triphosphate (Gpp(NH)p) was markedly affected. There was a significant increase in adenylyl cyclase activation by Gpp(NH)p in membranes from cells treated with low concentrations of TSH (less than or equal to 0.1 mU/ml) or forskolin (less than or equal to 1 microM) but a significant decrease in membranes from cells cultured with a higher concentration of TSH (1 mU/ml) or forskolin (10 microM). This decrease in Gpp(NH)p-stimulated adenylyl cyclase activity was mimicked by 8-bromo-cAMP but not by 1,9-dideoxyforskolin, a forskolin analogue which has lost its ability to activate adenylyl cyclase. There was a good correlation with the ability of Gs protein to be ADP-ribosylated by cholera toxin: labeling of Gs protein decreased following chronic treatment of thyroid cells with TSH (1 mU/ml) or forskolin (10 microM). In contrast, under the same experimental culture conditions a slight but significant increase in the quantity of Gs subunits was observed by immunoblotting analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Alkaline phosphatase prevents platelet stimulation by thromboxane-mimetics.

1. The effects of alkaline phosphatase on platelet aggregation, secretion and thromboxane B2 (TxB2) generation induced by the full dose-range of common platelet agonists were studied in human platelet-rich plasma and washed platelets. 2. Platelet aggregation and adenosine 5'-triphosphate (ATP) secretion induced by threshold and supramaximal concentrations of arachidonate and stable TxA2 and prostaglandin endoperoxide-mimetics (compounds U46619 and EP171) were abolished in the presence of alkaline phosphatase (0.5-1 u ml-1), even though the synthesis of TxB2 persisted. In contrast, platelet aggregation by PAF-acether and by supramaximal concentrations of thrombin as well as the primary wave of aggregation by adenosine diphosphate (ADP) and adrenaline were unaffected by alkaline phosphatase under conditions where the secondary wave of aggregation by ADP was blocked. 3. Alkaline phosphatase, unlike prostacyclin, failed to raise the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of the platelets. Also, the pretreatment of platelets by inorganic phosphate or by ATP plus creatine phosphate/creatine phosphokinase reversed the inhibitory effect of alkaline phosphatase. 4. Experiments performed in the guinea-pig in vivo showed that alkaline phosphatase was effective on thrombocytopenia induced by arachidonate. 5. Our results provide the first direct evidence for a specific inhibitory effect of alkaline phosphatase at a site sensitive to TxA2 and prostaglandin endoperoxides and suggest that its phosphorylation/dephosphorylation state may play an important role in modulating platelet activation. These results also suggest the presence of ecto-protein kinases on membrane platelets.

Alkaline Phosphatase↗

PubMed1991

Effects of pertussis toxin on the erythropoietin-stimulated proliferation and differentiation of erythroid-responsive cells.

Erythropoietin (Ep) stimulates the proliferation and differentiation of erythroid progenitor cells. We have investigated the possibility that guanyl nucleotide regulatory (G) proteins may be involved in Ep signal transduction. Pertussis toxin (PT) was found to inhibit Ep-stimulated [3H]-thymidine incorporation and large erythroid colony formation in a dose-dependent fashion, but had no effect on small colony formation or on Ep-induced differentiation. PT specifically adenosine diphosphate-ribosylated a major protein with an apparent molecular mass of 41 kD in erythroid progenitor cell membranes. These findings indicate that the transduction of the Ep signal for proliferation could be mediated in the early steps by a PT-sensitive G protein.

Animals↗

PubMed1990

Production of lipocortin-like proteins by cultured human tracheal submucosal gland cells.

Evidence is obtained for the presence of lipocortin-like proteins in human tracheal gland cells in culture. Using polyclonal antibodies to lipocortin I, indirect immunofluorescence studies demonstrate that lipocortin I is mainly confined to the tracheal gland cell surface. From cell membranes, four Ca2(+)-dependent proteins (35, 40, 45 and 67 kDa) were identified as lipocortin related proteins by using immunoblotting and fluorography following [35S]methionine metabolic labeling experiments. A strong immunoreactivity for the 35 kDa protein was observed. In addition, lipocortin-like proteins with apparent Mr33, 35, 37 and 67 kDa, respectively, were released in the apical culture medium by tracheal gland cells cultured on microporous membrane of a double chamber culture system.

Annexins↗

PubMed1990

Changes in cAMP-dependent and Ca2(+)-phospholipid-dependent protein kinase activities in suspension cultures of porcine thyroid cells.

The morphological and functional characteristics and the activities of cyclic AMP- (PKA I and PKA II) and calcium and phospholipid-dependent (PKC) protein kinases were studied in 2-day-old suspension cultures of porcine thyroid cells and were compared with those in freshly dissociated cells and intact glands. Thyroid cell morphology changed during the 2-day culture in the absence of specific regulators. This is characterized by a loss of cellular polarity, exo- and endocytotic vesicles and membranes of the rough endoplasmic reticulum, and an increase in the number of lysosomes, pseudomyelinic structures, lipidic inclusions and free ribosomes. Functional changes are characterized by a progressive decrease in protein iodination and its sensitivity to TSH stimulation. The total PKA activity in the cytosols of these cultures was slightly greater than that of freshly prepared tissue, due to the selective and significant accumulation of PKA I in cultured cells. In the particulate fraction the PKA activity was unchanged. PKC is the major kinase activity in porcine thyroids, and remains so in cultured cells. The slight drop in its activity in cytosols was offset by a significant increase in the particulate fraction, suggesting an intracellular redistribution of this kinase in cultured cells. The PKC activity is also partly activated in both the cytosol and particulate fraction, which results in an increased basal activity. The changes in PKA and PKC activities greatly modified the PKC/PKA ratios in the cytosols and the particulate fractions of cultured cells. These modifications could be partly responsible for the changes in sensitivity of cultured cells to the agents which control their activity.

Animals↗

PubMed1990

Control by thyrotropin of the production by thyroid cells of an inositol phosphate-glycan.

The increased turnover of phosphatidylinositol promoted by thyrotropin (TSH) in pig thyroid tissue does not seem to be caused by an increased production of inositol tris-phosphate. We have explored another possibility, the synthesis of an inositol phosphate-glycan (IP-gly). Our results show that thyroid cells in culture produced this substance from a precursor phosphatidylinositol-glycan (Gly-PI). The obtained IP-gly seemed, by its analytical and biological properties, to be identical, or similar, to the previously described insulin mediator.

Animals↗

PubMed1990

[Modifications of protein kinase C activity and phosphorylation of lipocortin I in cultures of pig thyroid cells].

When cultured in the absence of thyreostimulin (TSH), thyroid cells lose some of their differentiated functions such as iodide transport and its incorporation into thyroglobulin. In the presence of TSH (0.1 mU/ml), these differentiated functions are preserved ("TSH cells"). The addition of tetradecanoyl phorbol 13 acetate (TPA) inhibits some differentiated functions of the cells and provokes important modifications of bio-signalling pathways. The protein kinase C (pKC) activity, unchanged in "control" and "TSH cells", was dramatically modified in TPA treated cells. After translocation, the pKC activity was down-regulated and the phosphorylation of its endogenous substrates (35-38 kDa) disappeared. Among these substrates, we identified the lipocortin I (LC I) (35 kDa), a phospholipase A2 inhibitory protein related to the Ca2+ binding protein family. By monodimensional electrophoresis (PAGE-SDS) and western-blot, we evidenced the presence of LCI in cytosols and particulate extracts. By 2 dimensional electrophoresis (PAGE-SDS and IEF) and western-blot we identified a phosphorylated and unphosphorylated LCI protein. The phosphorylation of LCI by pKC decreased its isoelectric point from 6.9-6.6. The modifications of pKC activity and LCI phosphorylation and the changes in the bio-signalling pathways can partly account for the loss of differentiation observed in control or TPA treated cells.

Animals↗

PubMed1989

Identification of four lipocortin proteins and phosphorylation of lipocortin I by protein kinase C in cytosols of porcine thyroid cell cultures.

Four proteins of the lipocortin family, lipocortin I (35 kDa), lipocortin II (36 kDa), lipocortin V (32 kDa) and lipocortin VI (67-70 kDa), were identified in the cytosols of 2-day-old cultures of thyroid cells. Only lipocortin I was phosphorylated in vitro in fully differentiated, thyroid stimulating hormone-treated cells (0.1 mU/ml). Protein kinase C was the only kinase activity which phosphorylated lipocortin I. Phosphorylation shifted its pI from 6.9 to 6.6. The in vitro phosphorylation of lipocortin I was impaired in cultures exposed for 2 days to phorbol ester (10(-7) M), although it was present in both the cytosol and the particulate fraction of these cells.

Animals↗

PubMed1989

Effects of thyroid-stimulating hormone and phorbol ester on glycosaminoglycan synthesis in porcine thyroid epithelial cells in primary culture.

The effects of thyroid-stimulating hormone (TSH) and a tumor promoter: 12-0-tetradecanoyl-phorbol-13-acetate on glycosaminoglycan (GAG) synthesis were studied in porcine thyroid epithelial cells in primary culture. TSH is known to involve cyclic AMP mechanism and phorbol ester to act by protein kinase C pathway. Chronic treatment of cells with TSH increased the synthesis of heparan sulphate associated with the cell layer and hyaluronic acid in the culture medium. Phorbol ester increased the radioactivity of total GAGs in the culture medium but had no effect on GAGs associated with the cell layer. It inhibited the positive effect of TSH on heparan sulphate synthesis. These results suggest that in thyroid epithelial cells the synthesis of the GAGs associated with the cell layer and those secreted into the culture medium are regulated by different intracellular mechanisms.

Animals↗

PubMed1988

Purification and characterization of phospholipase A2 inhibitory proteins from pig thyroid gland.

A 32 kDa phospholipase A2 inhibitory protein was isolated from pig thyroid gland after calcium precipitation and fast protein liquid anion-exchange chromatography. SDS-polyacrylamide gel electrophoresis revealed the purity of the protein. The protein activity was assessed by the inhibition of pancreatic phospholipase A2 on [3H]oleic acid-labelled Escherichia coli membranes as substrate and on the prostaglandin E2 production of cultured thyroid cells. The amino acid composition and the isoelectric point were quite similar to those of endonexin previously described in other tissues or cells. The cross-reactivity of a polyclonal antibody against a 32 kDa lipocortin from human peripheral blood mononuclear cells with our thyroidal 32 kDa protein confirmed its lipocortin nature. Before the purification by fast protein liquid chromatography, the Ca2+ pellet contained lipocortin I (35 kDa and its core protein 33 kDa) identified by its cross-reactivity with a polyclonal antibody.

Amino Acids↗

PubMed1988

Phorbol ester prevents the thyroid-stimulating-hormone-induced but not the forskolin-induced decrease of cAMP-dependent protein kinase activity in thyroid cell cultures.

The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells. In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed Ca2+ and phospholipids for its activation. The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation. The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%). Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA. TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent. Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Activation of adenylate cyclase in rat fat cells promotes an increase in GTP content which controls the enzyme activity.

In previous studies, we demonstrated that the treatment of adipocytes with cholera toxin or Bordetella pertussis toxin (IAP) promoted an increase in the total guanosine triphosphate (GTP) content of the cells concomitant with the increase in cyclic adenosine monophosphate (AMP) level and the resulting lipolysis. In the present studies, we show that the acute challenge of fat cells with 1 microM isoproterenol (IPNE) is associated with a transient increase in GTP level (3-fold in 6 min). This increase may be attributed to an inhibition of the disposal of GTP or to a stimulation of its synthesis. To evaluate the actual role of GTP, we used virazole, an antitumor agent which inhibits inosinic acid dehydrogenase. After 2 h preincubation of the cells with 1 mM virazole, the effect of a 6 min challenge with 1 microM IPNE is decreased by 59% at the GTP level and by 42% in cyclic AMP production. One hour later, the resulting lipolytic efficiency is reduced by 57%. IAP treatment (10 micrograms/ml) produced its maximal effect on GTP and cyclic AMP levels and on lipolysis after 90 min incubation. The antilipolytic effect of 1 microM phenylisopropyladenosine (PIA) is almost abolished. When 1 mM virazole is added to the cell suspension to deplete the guanyl nucleotide pool, the resulting lipolysis due to IAP treatment is decreased by 85%, whereas GTP and cyclic AMP levels were decreased by 80 and 70%, respectively. We can conclude that the cyclic AMP synthesis in intact cells is accompanied by a parallel increase of their GTP content, whether the stimulation results from the activation of Gs or the inhibition of Gi. The reduction of the guanyl nucleotide pool under virazole results in a relatively less important inhibition of lipolysis when Gs is stimulated than when it is Gi.

Adenosine Triphosphate↗

PubMed1988

Decrease in cAMP-dependent protein kinase activity in suspension cultures of porcine thyroid cells exposed to TSH or forskolin.

Suspension cultures of porcine thyroid cells were used to study the action of TSH and forskolin (Fk) on cAMP-dependent (PKa) and Ca2+-phospholipid-dependent (PKc) protein kinase--enzymes which represent the key step in the transduction of extracellular signals. The PKa activity in cells cultured for 2 days in the presence of TSH was decreased to about 50% of control level with a TSH dose of 0.1 mU/ml. This decrease is dose dependent; only traces of PKa activity remained at very high doses of TSH (50 mU/ml). Similar results were obtained with Fk (10(-5) M), the adenylate cyclase activator. It decreased the PKa activity to the level obtained with 0.1-1.0 mU/ml TSH. The loss of the PKa activity was parallel in cytosol and particulate fractions, suggesting that there is no translocation of enzymes under the action of either TSH or Fk. Neither TSH nor Fk had any effect on PKc, which became the predominant activity in cells exposed to either of the regulators. The cAMP-dependent phosphorylation of endogenous proteins was lower in TSH- or Fk-treated cells than in controls, and was dependent, like the PKa activity, on the dose of TSH. Polyacrylamide gel electrophoresis (PAGE) revealed the specific substrates of PKa in cultured thyroid cells. Proteins of 28, 30 and 33 kDa were regularly found, while 58 kDa protein was not present in all experiments. PAGE patterns showed that the decrease in endogenous phosphorylation in TSH- and Fk-treated cells was due to decreased labelling of PKa-specific substrates. The observed down-regulation of PKa activity could have an influence on the expression of thyroid cell differentiation.

Animals↗

PubMed1986

Modulation by cyclic AMP of arachidonic acid-induced platelet desensitization.

We have previously demonstrated that arachidonic acid (AA) and the stable cyclic endoperoxide analogue (U46619) desensitize human platelets at a common site, which is sensitive to endoperoxides/thromboxane receptor antagonists. We now report on the influence of agents which evaluate intracellular levels of platelet adenosine 3',5'-cyclic monophosphate (cAMP) on AA- and U46619-induced platelet desensitization. Prostaglandin E1, prostacyclin, carbacyclin, forskolin or dibutyryl cAMP prevented platelet activation by and desensitization to AA and to U46619 under conditions where the formation of thromboxane B2 was not significantly modified. Inhibition of platelet activation (aggregation and secretion) required a lower increase of the cAMP content than was needed to inhibit desensitization, confirming previous findings that desensitization to and by AA or U46619 are independent from the platelet release reaction. Together, these results indicate that AA-induced desensitization can be modulated by the adenylate cyclase/cAMP system acting at a site distinct from the known mechanisms of Ca2+ sequestration. This site is shared by the AA metabolite responsible for desensitization and by U46619 and is related to their common platelet membrane receptor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1986

Relationships between the GTP content and the TSH-stimulated adenylate cyclase activity of cultured thyroid cells.

The adenylate cyclase activity of a crude membrane fraction derived from cells cultured for 4 days in the presence of TSH (0.1 mU/ml), when acutely stimulated with 25 mU/ml, is 5-8 times higher than that derived from control cells. It has been suggested that changes in the intracellular content of GTP resulting from TSH chronic treatment were the cause of the modified responsiveness of the cyclase. To investigate this hypothesis, a method for GTP determination was developed. The steady-state concentration of GTP in 4-day TSH-treated cells is 2-3 times higher than in 4-day control cells. The increase in GTP content is concentration dependent between 5 and 500 microU/ml TSH in the culture medium. It presents a maximum on day 4 of culture, but remains elevated up to day 5. Nevertheless the GTP content is not the only factor controlling the cyclase activity, indeed the addition of 0.1 mM GTP to membranes from control cells does not increase the response up to the level reached by membranes from TSH-treated cells. Treatment of the cells with virazole, a drug inhibiting the biosynthesis of guanyl nucleotides, greatly decreases the GTP level, but is unable to suppress the positive effect of the TSH chronic treatment on adenylate cyclase activity. These results show that the increase in GTP level resulting from culture of the cells in the presence of minute amounts of TSH is not exclusively linked to adenylate cyclase responsiveness to TSH.

Adenosine Triphosphate↗