PubMed Health⌕ Search

Biomedical subjects

D Corda

Publications and source records attributed to D Corda.

At least 55 records · Page 3Linked to original sources
PubMed1994

Elevated levels and mitogenic activity of lysophosphatidylinositol in k-ras-transformed epithelial cells.

In cell lines stably (KiKi) or reversibly (Ts) transformed by the k-ras oncogene originated from a differentiated rat thyroid line (FRTL5 cells), k-ras-induced transformation has been associated with an increased phospholipase A2 activity. Here we provide evidence that this enzymic activity is phosphoinositide specific and leads to the formation of lysophosphatidylinositol. The levels of this lysolipid increased by 2-3-fold in ras-transformed cells (KiKi cells and Ts cells at the permissive temperature of 33 degrees C) as compared to differentiated cells (FRTL5) or to Ts cells maintained at 39 degrees C, i.e. at the temperature where ras-p21, the product of the ras oncogene, is inactive. Since another lysoderivative, lysophosphatidic acid, has been shown to be a mitogen, we have tested whether lysophosphatidylinositol could have a similar activity on thyroid cells. Lysophosphatidylinositol (10-100 microM) induced a 5-10-fold increase in [3H]thymidine incorporation in both FRTL5 and KiKi cells, whereas lysophosphatidic acid was active only in differentiated cells. Lysophosphatidylinositol (approximately 25 microM) and lysophosphatidic acid (50-100 microM) acted synergistically with insulin in increasing [3H]thymidine incorporation. Moreover, lysophosphatidylinositol at concentrations three-fold higher than those found to be mitogenic, inhibited the activity of the GTPase-activating protein. We conclude that lysophosphatidylinositol is a mitogen that might play a role in the modulation of k-ras transformed cell proliferation.

Animals↗
PubMed1994

Stimulation of endogenous ADP-ribosylation by brefeldin A.

Brefeldin A (BFA) is a fungal metabolite that exerts profound and generally inhibitory actions on membrane transport. At least some of the BFA effects are due to inhibition of the GDP-GTP exchange on the ADP-ribosylation factor (ARF) catalyzed by membrane protein(s). ARF activation is likely to be a key event in the association of non-clathrin coat components, including ARF itself, onto transport organelles. ARF, in addition to participating in membrane transport, is known to function as a cofactor in the enzymatic activity of cholera toxin, a bacterial ADP-ribosyltransferase. In this study we have examined whether BFA, in addition to inhibiting membrane transport, might affect endogenous ADP-ribosylation in eukaryotic cells. Two cytosolic proteins of 38 and 50 kDa were enzymatically ADP-ribosylated in the presence of BFA in cellular extracts. The 38-kDa substrate was tentatively identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. The BFA-binding components mediating inhibition of membrane traffic and stimulation of ADP-ribosylation appear to have the same ligand specificity. These data demonstrate the existence of a BFA-sensitive mono(ADP-ribosyl)transferase that may play a role in membrane movements.

ADP Ribose Transferases↗
PubMed1994

Norepinephrine, unlike ATP, induces all-or-none increase in cytosolic calcium in thyroid cells. The role of inositol-trisphosphate-sensitive stores and calcium channels.

The mechanism of action of norepinephrine and ATP has been analyzed in single FRTL5 cells (a normal thyroid cell line), loaded with the fluorescent Ca2+ probe Fura2. ATP increased the cytosolic Ca2+ in an apparently concentration-dependent manner with a maximal effect at 10 microM (413 +/- 26% over basal levels of 135 +/- 7 nM). In contrast, the norepinephrine-induced increase (198 +/- 5% over basal) was concentration independent in individual cells, the minimal effective concentration being 1 nM. However, the number of cells responding to norepinephrine was concentration dependent. The ATP-induced Ca2+ rise was biphasic, consisting of a rapid rise (2-4 s, 252 +/- 15%), resembling the effect of norepinephrine, followed by a slower and longer component, which reached a plateau in 0.5-2 min. The second component appeared to be related to the opening of a channel, since it required extracellular Ca2+ and was abolished by SC38249, an inhibitor of the second-messenger-operated and voltage-operated channels. Moreover, it was inhibited by 4 beta-phorbol 12-myristate 13-acetate, suggesting that protein kinase C might be involved in the modulation of this Ca2+ channel.

Adenosine Triphosphate↗
PubMed1993

Glycerophosphoinositol 4-phosphate, a putative endogenous inhibitor of adenylylcyclase.

In a continuous line of rat thyroid cells transformed by the k-ras oncogene (KiKi), the expression of ras-p21 correlates with an increased activity of a phosphoinositide-specific phospholipase A2, which leads to elevated levels of glycerophosphoinositols. In this study we have characterized the biological activities of these compounds. Growth and differentiation in thyroid cells are mainly regulated by the activation of adenylylcyclase. Therefore, we have studied the effects of glycerophosphoinositols on the activity of this enzyme using a normal thyroid cell line (FRTL5). Micromolar concentrations of glycerophosphoinositol 4-phosphate (GroPIns-4-P) caused a approximately 50% inhibition of the adenylylcyclase activity in FRTL5 membranes stimulated by the GTP-binding protein activator fluoroaluminate. Similar concentrations of GroPIns-4-P were detected in KiKi cells but not in the normal FRTL5 line. Micromolar GroPIns-4-P was found to be taken up by intact FRTL5 cells and to induce nearly 50% inhibition of the thyrotropin- and cholera toxin-induced increase in cAMP levels. Similar results were also observed in other cell lines (smooth muscle, pituitary cells, and pneumocytes). GroPIns-4-P inhibited cAMP-dependent cellular functions such as iodide uptake and thymidine incorporation in FRTL5 cells when stimulated by thyrotropin and cholera toxin but not when induced by forskolin. These results are consistent with GroPIns-4-P exerting an inhibitory effect on the GTP-binding protein that stimulates adenylycyclase. We propose that GroPIns-4-P might mediate a mechanism of cross-talk between adenylylcyclase and phospholipase A2 in thyroid as well as in other cell systems.

Adenosine Diphosphate Ribose↗
PubMed1993

How fish power swimming.

It is thought that fish generate the power needed for steady swimming with their anterior musculature, whereas the posterior musculature only transmits forces to the tail and does negative work. Isolated red muscle bundles driven through the length changes and stimulation pattern that muscles normally undergo during steady swimming showed the opposite pattern. Most of the power for swimming came from muscle in the posterior region of the fish, and relatively little came from the anterior musculature. In addition, the contractile properties of the muscle along the length of the fish are significantly adapted to enhance power generation.

Animals↗
PubMed1992

K-ras transformation greatly increases the toxin-dependent ADP-ribosylation of GTP binding proteins in thyroid cells. Involvement of an inhibitor of the ADP-ribosylation reaction.

The GTP binding (G) proteins of normal (FRTL5) and ras-transformed thyroid cells (KiKi) were characterized by cholera and pertussis toxin-induced ADP-ribosylation and immunoblot analysis. Two pertussis toxin substrates with molecular masses of 40 and 41 kDa were identified in normal cells as the alpha i2 and alpha i3 subunits. The molecular masses of the cholera toxin substrates were 42 and 45 kDa. The same cholera and pertussis toxin substrates were present in the K-ras-transformed cell line. However, the toxin-dependent ADP-ribosylation was markedly higher in KiKi than in normal cell membranes (more than 50-fold). The reason for this difference was investigated; it could not be explained by the relative amounts of G proteins in the two cell systems, since the levels of alpha i2 subunit as measured by quantitative immunoblot in K-ras-transformed cells were only slightly (65%) higher than in normal cells. The difference in ADP-ribosylation was not due to poly-ADP-ribosylation nor to a different degree of subunit dissociation of G proteins in the two cell lines. Rather, the enhanced ADP-ribosylation in K-ras-transformed cells appears to be due to the loss of an inhibitory factor present in the normal cells. Partial characterization indicates that such a factor is a peripheral membrane protein of less than 25 kDa capable of directly interfering with the ADP-ribosylation reaction.

Adenosine Diphosphate Ribose↗
PubMed1992

Immunoglobulins from Graves' patients stimulate phospholipase-A2 in FRTL5 thyroid cells.

The well documented ability of immunoglobulins G (IgGs) from Graves' patients to stimulate cAMP production is believed to be involved in the pathophysiology of this disease. It is still under discussion whether other intracellular messengers known to regulate thyroid function might play a similar role. This study shows that phospholipase-A2, a signal pathway unrelated to cAMP, is activated by Graves' IgGs. The IgGs from 67 patients with active Graves' disease, 8 patients with Graves' disease in remission, 5 patients with idiopathic myxedema, 2 patients with Hashimoto's thyroiditis, 57 patients with nonautoimmune thyroid disease, and 65 normal subjects were tested for their ability to stimulate phospholipase-A2 activity, as measured by arachidonic acid release from FRTL5 thyroid cells. The IgGs from patients with active Graves' disease caused a significant increase in arachidonic acid release compared to those from normal subjects, patients with nonautoimmune thyroid diseases, and patients with Graves' disease in remission (P less than 0.0001). The IgGs from active Graves' patients were also able to increase cAMP accumulation in FRTL5 cells. This effect did not correlate with the ability of the same IgGs to induce arachidonic acid release, suggesting that Graves' IgGs stimulate these two pathways by separate mechanisms. Moreover, a subgroup of IgGs that stimulated phospholipase-A2 did not increase the cAMP levels in FRTL5 cells. Our data suggest a novel mechanism of action of Graves' IgGs, the activation of phospholipase-A2, well distinguishable from the known effect on cAMP accumulation. The assay we describe could be helpful in improving the diagnosis and therapy of Graves' disease and in distinguishing it from nonautoimmune thyroid diseases. It also supplies the basis for a prospective subclassification of the Graves' patients, which might become useful to clarify the pathophysiology of this disease.

Adolescent↗
PubMed1991

Transformation by the k-ras oncogene correlates with increases in phospholipase A2 activity, glycerophosphoinositol production and phosphoinositide synthesis in thyroid cells.

Two cell lines transformed by the k-ras oncogene (KiKi and KiMol cells) and a temperature sensitive clone (Ts), all originated from a normal rat thyroid line (FRTL5 cells), have been employed to analyse the intracellular mechanisms affected by the ras p21. In k-ras transformed cells two phosphoinositide derivatives, glycerophosphoinositol and inositol monophosphate, were markedly increased, whereas inositol bisphosphate and trisphosphate maintained the same level as in normal cells. Cytosolic Ca2+ was also unaffected. This indicates that in epithelial cells the phospholipase C activity is not altered upon ras transformation. The formation of glycerophosphoinositol involved the activation of a phosphoinositide specific phospholipase A2. The higher phospholipase A2 activity in ras transformed cells could be further demonstrated by the increase in total arachidonic acid release. In the Ts clone the increase in glycerophosphoinositol and inositol monophosphate was evident only at the permissive temperature (33 degrees C), whereas it disappeared at 39 degrees C. At 33 degrees C the cells were also characterized by an enriched membrane pool of phosphoinositides. All these changes occurred in parallel with morphological transformation. We propose that cell transformation by the k-ras oncogene affects different steps of the membrane lipid metabolism, among which the most prominent one is the activation of a phosphoinositide specific phospholipase A2. These effects could originate mitogenic metabolites. Moreover, they correlate well with the induction of the malignant phenotype.

Animals↗
PubMed1991

Muscarinic regulation of phospholipase A2 and iodide fluxes in FRTL-5 thyroid cells.

FRTL-5 thyroid cells express a muscarinic receptor which inhibits the phospholipase C activity in a pirenzepine-insensitive manner. We here report that the cholinergic agonist carbachol decreases in these cells the steady-state iodide content, an effect correlated with the iodination of thyroglobulin and with thyroid hormone formation. Several signal pathways may be involved in this phenomenon since carbachol in addition to inhibiting phospholipase C, increased the arachidonic acid release and modified the adenylyl cyclase activity. In FRTL-5 cells, arachidonic acid is released via the direct stimulation of phospholipase A2 by a pirenzepine-sensitive muscarinic receptor coupled to a GTP binding protein sensitive to pertussis toxin. Regarding adenylyl cyclase, carbachol potentiated the thyrotropin-induced stimulation of the enzyme, whereas it did not affect the basal levels of cAMP. In vitro binding studies revealed the presence of two muscarinic binding sites. To summarize, the analysis of signal pathways and of in vitro binding sites indicates a complex muscarinic regulation of thyroid function, which includes the modulation of iodide fluxes.

Animals↗
PubMed1990

Evidence that a guanine nucleotide-binding protein linked to a muscarinic receptor inhibits directly phospholipase C.

The mechanism of phospholipase C regulation by inhibitory receptors was analyzed both in intact and in permeabilized rat thyroid cells (FRTL5). In this system, the muscarinic agonist carbachol inhibited phospholipase C, as indicated by the decrease in the basal levels of inositol 1,4,5-trisphosphate as well as by the reduced adrenergic stimulation of phosphoinositol accumulation, which was paralleled by a fall in the cytosolic Ca2+ levels. This inhibition involved an M2 muscarinic receptor because it was abolished by atropine but not by the M1 antagonist pirenzepine. Cells pretreated with pertussis toxin were not responsive to carbachol, indicating the involvement of a guanine nucleotide-binding protein in this inhibitory process. This possibility was further evaluated in permeabilized cells, where the carbachol inhibition was shown to be completely dependent on GTP. Known second messengers were not involved in this inhibitory process since Ca2+, cAMP, and activators of protein kinases were not able to mimic or prevent the carbachol effect either in intact or in permeabilized FRTL5 cells. In this system, the phospholipases C and A2 are coupled to two classes of muscarinic receptors that display a different sensitivity to pertussis toxin. The carbachol inhibitory effect occurred under conditions that prevented activation of phospholipase A2, excluding a role of the arachidonic acid metabolism in this process. Taken together these data provide the strongest support to date that an inhibitory guanine nucleotide-binding protein sensitive to pertussis toxin can directly mediate receptor-induced inhibition of phospholipase C.

Alkaloids↗
PubMed1989

G protein-linked receptors in the thyroid.

The FRTL5 cell line has the advantage that its hormonal activation leads to important and measurable thyroid function such as the transport of iodide and the iodination of thyroglobulin. Secondly, the coexistence in the plasma membrane of these cells of several physiologically relevant receptors (TSH, alpha 1-adrenergic, M1 and M2 muscarinic, insulin, IGF1) coupled to at least three transducing enzymes (adenylyl cyclase, PLC, PLA2) gives the possibility to analyze the interaction among second messengers in the cell activation process. This has allowed us and others to show that in the case of the iodide efflux regulation at least two second messengers (Ca++ and arachidonic acid) mediate the adrenergic stimulation, whereas the TSH activation of the same phenomenon probably uses other signals in addition to Ca++ and arachidonic acid. Growth is mostly regulated by TSH, that activates the adenylyl cyclase by a mechanism that may involve the modulation of the availability of Gi. TSH is also able to regulate an endogenous ADP ribosyl transferase in FRTL5. This could be a novel mechanism of cell regulation by this hormone, but the role of this phenomenon in the physiological action of TSH is still unclear.

Adenylyl Cyclases↗
PubMed1989

Changes in the synthesis of histone H1(0) and H1 in rat FRTL-5 thyroid cells exposed to thyrotropin.

In absence of thyrotropin (TSH), FRTL-5 rat thyroid cells stop proliferating and lose the functional characteristics of thyroid tissue. FRTL-5 cells regain their differentiated state and their proliferation activity upon addition of TSH. In this study we investigated the synthesis of histone H1 variants and H19(0) in FRTL-5 cells exposed to 10(-8) M TSH, two days after TSH withdrawal. TSH induced the synthesis of some H1 variants and H1. This effect was already evident six hours after TSH addition, thus well before proliferation, DNA or thyroglobulin synthesis was induced. These data indicate that the induction of H1(0) and some H1 variants is an early event after TSH stimulation and may thus be related to the functional differentiation of FRTL-5 cells.

Animals↗
PubMed1988

Adenylate cyclase activity of v-ras-k transformed rat epithelial thyroid cells.

The regulation of adenylate cyclase has been analyzed in normal rat thyroid cells as well as in the same cells transformed by the v-ras-k oncogene. In both cell types the adenylate cyclase complex consists of the two GTP-binding proteins, Gi and Gs, as demonstrated by the specific ADP-ribosylation induced by pertussis and cholera toxin, respectively. The response of adenylate cyclase of the transformed cells to forskolin, pertussis toxin and cholera toxin is attenuated with respect to the control cell line. The thyrotropic hormone (TSH), that acts on normal thyroid cells in culture as a growth factor by stimulating the adenylate cyclase activity, is not able to induce DNA synthesis nor does it stimulate adenylate cyclase in v-ras-k transformed cells.

Adenylate Cyclase Toxin↗
PubMed1987

Thyrotropin effect on the availability of Ni regulatory protein in FRTL-5 rat thyroid cells to ADP-ribosylation by pertussis toxin.

Incubation of FRTL-5 rat thyroid cell membranes with [32P]NAD and pertussis toxin results in the specific ADP-ribosylation of a protein of about 40 kDa. This protein has the same molecular mass of the alpha i subunit of the adenylate cyclase regulatory protein Ni and is distinct from proteins ADP-ribosylated by cholera toxin in the same membranes. Prior treatment of FRTL-5 cells with pertussis toxin results in the ADP-ribosylation of Ni, as indicated by the loss of the toxin substrate in the ADP-ribosylation assay performed with membranes prepared from such cells. Preincubation of FRTL-5 cells with thyrotropin causes the same loss; cholera toxin has no such effect. Pertussis toxin, as do thyrotropin and cholera toxin, increases cAMP levels in FRTL-5 cells. Forskolin together with thyrotropin, cholera toxin or pertussis toxin causes a further increase in cAMP levels. Pertussis toxin and thyrotropin are not additive in their ability to increase adenylate cyclase activity, whereas both substances are additive with cholera toxin. A role of Ni in the thyrotropin regulation of the adenylate cyclase activity in thyroid cells is proposed.

Adenosine Diphosphate Ribose↗
PubMed1987

Phorbol myristate acetate inhibits alpha 1-adrenergically but not thyrotropin-regulated functions in FRTL-5 rat thyroid cells.

The iodination of thyroglobulin and the formation of thyroid hormones are regulated by alpha 1-adrenergic agents as well as TSH in rat FRTL-5 cells. The regulatory effects of the alpha-1-adrenergic agents and TSH on both of these processes are associated with an increase in cytosolic Ca2+ and an increase in that component of iodide efflux that is representative of the movement of iodide from the thyroid cell into the follicular lumen. When FRTL-5 cells are preincubated with phorbol myristate acetate (PMA) for at least 3 min, the norepinephrine-stimulated changes in cytosolic Ca2+ levels and iodide efflux are inhibited. In contrast, PMA pretreatment has no effect on iodide efflux and actually enhances the changes in cytosolic Ca2+ induced by TSH. Phorbol myristate acetate pretreatment has no effect on TSH-stimulated cAMP-mediated iodide uptake in FRTL-5 cells, nor does it affect the binding parameters of the alpha 1-adrenergic receptor antagonist prazosin. These data suggest that protein kinase C is involved in a feedback mechanism regulating alpha 1-adrenergic but not TSH-induced changes associated with the iodination of thyroglobulin and the formation of thyroid hormones; and that this feedback effect occurs after the step of ligand binding but before the increase in cytosolic Ca2+ induced by the alpha 1-adrenergic agents.

Animals↗
PubMed1986

Role of pertussis toxin sensitive G proteins in the alpha 1 adrenergic receptor but not in the thyrotropin receptor mediated activation of membrane phospholipases and iodide fluxes in FRTL-5 thyroid cells.

The regulation of thyroid hormone formation by thyrotropin and norepinephrine involves the activation of both phospholipases C and A2. When FRTL-5 cells are incubated with 10(-10)M pertussis toxin for 4 to 20 h, the stimulation of iodide efflux by norepinephrine is inhibited by 50 to 70%. At the same toxin concentration the norepinephrine induced increase in cytosolic Ca2+ is unaffected; however upon 20 h pretreatment with 10(-9)M pertussis toxin a 30% inhibition is observed. By contrast, the pertussis toxin treatment had no effect on the increase in iodide efflux or in cytosolic Ca2+ levels induced by thyrotropin. Our data suggest that two GTP binding proteins sensitive to pertussis toxin are involved in the alpha 1 adrenergic but not in the thyrotropin induced activation of the signal transduction mechanisms leading to iodide efflux in FRTL-5 cells.

Animals↗