Thyroid hormone inhibition in L6 myoblasts of IGF-I-mediated glucose uptake and proliferation: new roles for integrin αvβ3.

Thyroid hormones L-thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3) have been shown to initiate short- and long-term effects via a plasma membrane receptor site located on integrin αvβ3. Also insulin-like growth factor type I (IGF-I) activity is known to be subject to regulation by this integrin. To investigate the possible cross-talk between T4 and IGF-I in rat L6 myoblasts, we have examined integrin αvβ3-mediated modulatory actions of T4 on glucose uptake, measured through carrier-mediated 2-deoxy-[3H]-D-glucose uptake, and on cell proliferation stimulated by IGF-I, assessed by cell counting, [3H]-thymidine incorporation, and fluorescence-activated cell sorting analysis.

Effect of atrial natriuretic peptide on reactive oxygen species-induced by hydrogen peroxide in THP-1 monocytes: role in cell growth, migration and cytokine release.

Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. The aim of the present study was to investigate in THP-1 monocytes the ANP effect on hydrogen peroxide (H2O2)-induced Reactive Oxygen Species (ROS), cell proliferation and migration. A significant increase of H2O2-dependent ROS production was induced by physiological concentration of ANP (10(-10)M).

Protection of cells against oxidative stress by nanomolar levels of hydroxyflavones indicates a new type of intracellular antioxidant mechanism.

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide.

Sticholysin II: a pore-forming toxin as a probe to recognize sphingomyelin in artificial and cellular membranes.

Sphingomyelin is a major component of membrane rafts, and also is a precursor of many bioactive molecules. The sphingomyelin plays important biological roles and alterations of its metabolism are the basis of some genetic disorders such as the Niemann Pick disease. A complete understanding of its biological role is frustrated by the lack of efficient tools for its recognition in the cell.

Nongenomic effects of thyroid hormones on the immune system cells: New targets, old players.

It is now widely accepted that thyroid hormones, l-thyroxine (T(4)) and 3,3',5-triiodo-l-thyronine (T(3)), act as modulators of the immune response. Immune functions such as chemotaxis, phagocytosis, generation of reactive oxygen species, and cytokine synthesis and release, are altered in hypo- and hyper-thyroid conditions, even though for many immune cells no clear correlation has been found between altered levels of T(3) or T(4) and effects on the immune responses. Integrins are extracellular matrix proteins that are important modulators of many cellular responses, and the integrin αvβ3 has been identified as a cell surface receptor for thyroid hormones.

Thyroid hormones as modulators of immune activities at the cellular level.

Background: Increasing evidence suggests that thyroid hormones, L-thyroxine (T(4)) and 3,3',5-triiodo-L-thyronine (T(3)), are modulators of the immune response. In monocytes, macrophages, leukocytes, natural killer cells, and lymphocytes, a wide range of immune functions such as chemotaxis, phagocytosis, generation of reactive oxygen species (ROS), and cytokine synthesis and release are altered under hypo- and hyperthyroid conditions.

Summary: Hyperthyroidism decreases the proinflammatory activities of monocytes and macrophages, whereas enhancement of phagocytosis and increased levels of ROS may occur during hypothyroidism.

Isolation and characterization of a myotoxin from the venom of Macrovipera lebetina transmediterranea.

The Macrovipera lebetina venom consists of a complex mixture of proteins belonging to a few main families according to their enzymatic and pharmacological activity. Given the serious pathophysiological effects caused by M. lebetina bites mainly induced by muscle degeneration, we decided to investigate the myotoxic activity of some venom fractions.

Atrial natriuretic peptide and oxidative stress.

Atrial natriuretic peptide (ANP) is a hormone, produced mainly by cardiomyocytes, with a major role in cardiovascular homeostatic mechanisms such as natriuresis and vasodilation, which serve to regulate blood pressure. However, ANP also acts as an autocrine/paracrine factor on other targets such as kidney, lung, thymus, liver and the immune system. ANP participates in the regulation of cell growth and proliferation, and evidence is accumulating that these effects are associated with the generation of reactive oxygen species (ROS).

P-Glycoprotein is not a key target for the chemosensitizing effect of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol in HepG2 cells exposed to doxorubicin.

Chemosensitization of HepG2 cells to doxorubicin by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol neither impinged on downregulation of P-glycoprotein expression nor on severe impairment of its activity. Moreover, differently from verapamil, a potent P-glycoprotein inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitized HepG2 cells in a fashion that was insensitive to the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. At concentrations exceeding the one employed for chemosensitization, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol was by itself strongly toxic to HepG2 cells, and also this effect was insensitive to the pancaspase inhibitor.

Short-term effects of thyroid hormones during development: Focus on signal transduction.

Extranuclear or nongenomic effects of thyroid hormones are mediated by receptors located at the plasma membrane or inside cells, and are independent of protein synthesis. Recently the alphaVbeta3 integrin was identified as a cell membrane receptor for thyroid hormones, and a wide variety of nongenomic effects have now been shown to be induced through binding of thyroid hormones to this receptor. However, also other thyroid hormone receptors can produce nongenomic effects, including the cytoplasmic TRalpha and TRbeta receptors and probably also a G protein-coupled membrane receptor, and increasing importance is now given to thyroid hormone metabolites like 3,5-diiodothyronine and reverse T(3) that can mimick some nongenomic effects of T(3) and T(4).

Cholesterol depletion inhibits electrophysiological changes induced by anoxia in CA1 region of rat hippocampal slices.

The hyper-activation of glutamate receptors is a key event in the degenerative processes triggered by ischemia in the brain. Several types of these receptors reside in cholesterol-sphingomyelin rich domains of post-synaptic plasma membranes and have been described to be sensitive to cholesterol depletion. Hence we investigated, by extracellular recordings, the effect of cholesterol depletion on population spikes (PS) during ischemia-like conditions in the CA1 region of rat hippocampal slices using the cholesterol-depleting agent methyl-beta-cyclodextrin (MbetaCD).

Oxidant-induced pHi/Ca2+ changes in rat aortic smooth muscle cells. The role of atrial natriuretic peptide.

The aim of this study was to investigate the effects of oxidative stress on PLD activity, [Ca2+]i and pHi levels and the possible relationship among them. Moreover, since atrial natriuretic peptide (ANP) protects against oxidant-induced injury, we investigated the potential protective role of the hormone in rat aortic smooth muscle (RASM) cells exposed to oxidative stress. Water-soluble 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) was used as free radical generating system, since it generates peroxyl radicals with defined reaction and the half time of peroxyl radicals is longer than other ROS.

Involvement of phospholipids in the mechanism of insulin action in HEPG2 cells.

The mechanism of action by which insulin increases phosphatidic acid (PA) and diacylglycerol (DAG) levels was investigated in cultured hepatoma cells (HEPG2). Insulin stimulated phosphatidylcholine (PC) and phosphatidyl-inositol (PI) degradation through the activation of specific phospholipases C (PLC). The DAG increase appears to be biphasic.

Differential sensitivity of human monocytes and macrophages to ANP: a role of intracellular pH on reactive oxygen species production through the phospholipase involvement.

Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. The aim of the present work was to study the effect of ANP on the intracellular pH (pHi) of human monocytes and macrophages and to investigate whether pHi changes could play a role on phospholipase activities and reactive oxygen species (ROS) production. Human macrophages isolated by peripheral blood mononuclear cells and THP-1 monocytes, which were shown to express all three natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C), were treated with physiological concentrations of ANP.

Atrial natriuretic factor inhibits mitogen-induced growth in aortic smooth muscle cells.

Atrial natriuretic factor (ANF) is a polypeptide able to affect cardiovascular homeostasis exhibiting diuretic, natriuretic, and vasorelaxant activities. ANF shows antimitogenic effects in different cell types acting through R(2) receptor. Excessive proliferation of smooth muscle cells is a common phenomenon in diseases such as atherosclerosis, but the role of growth factors in the mechanism which modulate this process has yet to be clarified.

Short-term effects of thyroid hormones and 3,5-diiodothyronine on membrane transport systems in chick embryo hepatocytes.

Rapid nongenomic effects of thyroid hormones L-T(3) and L-T(4) on two plasma membrane transport systems were investigated in 14-d-old and 19-d-old chick embryo hepatocytes. The Na(+)/H(+) exchanger activity was measured using the intracellular pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, whereas the amino acid transport was estimated by [1-(14)C]-2-aminoisobutyric acid uptake. System A amino acid transport activation was linear to hormone concentration, whereas the Na/H exchanger gave a bell-shaped dose-response curve, with a maximum at the physiological hormone concentration of 1 nM.

PLA(2) stimulation of Na(+)/H(+) antiport and proliferation in rat aortic smooth muscle cells.

The proliferative properties and the ability to stimulate the Na(+)/H(+) antiport activity of a secretory phospholipase A(2) were studied in rat aortic smooth muscle cells in culture. The requirement of the enzymatic activity of phospholipase A(2) to elicit mitogenesis was assessed by the use of ammodytin L, a Ser(49) phospholipase A(2) from the venom of Vipera ammodytes, devoid of hydrolytic activity. We propose that the proliferative effect is mediated by the same transduction pathway for both proteins.

Short-term effects of thyroid hormones on the Na/H antiport in L-6 myoblasts: high molecular specificity for 3,3',5-triiodo-L-thyronine.

The thyroid hormones L-T3 and L-T4 were shown to activate the Na/H antiport in L-6 cells from rat skeletal muscle by a rapid, nongenomic mechanism. Under pH equilibrium conditions, a significant rise in the intracellular pH, measured by the fluorescent pH indicator 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein was observed after the addition of physiological concentrations (10(-10) M) of either L-T3 or L-T4, but with different time courses. L-T3 at all concentrations increased the pH after a delay of 2 min, whereas L-T4 showed a concentration-dependent lag time, going from 11 min at 10(-11) M down to 5 min for a hormone concentration of 10(-6) M.

Dual modulation of Na/H antiport by atrial natriuretic factor in rat aortic smooth muscle cells.

The aim of the present work was to study the effect of the atrial natriuretic factor (ANF) on the Na/H antiport in rat aorta smooth muscle cells, evaluated as intracellular pH (pHi) recovery after an acid load with ammonium chloride. The Na/H antiport was studied using a fluorescent probe, sensitive to pHi, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Our data indicate that ANF modulates the activity of the Na/H antiport in both a dose- and time-dependent manner.

Insulin stimulation of Na/H antiport in L-6 cells: a different mechanism in myoblasts and myotubes.

Insulin modulation of the Na/H antiport of L-6 cells, from rat skeletal muscle was studied in both myoblasts and myotubes using the fluorescent, pH sensitive, intracellular probe 2',7' bis (carboxyethyl)-5(6)-carboxyfluorescein. Insulin stimulated the Na/H antiport activity in L-6 cells, showing a bell-shaped dose response typical of other insulin responses: a maximum at 10 nM (delta pH of 0.132 +/- 0.

Phosphatidylinositol- and phosphatidylcholine-dependent phospholipases C are involved in the mechanism of action of atrial natriuretic factor in cultured rat aortic smooth muscle cells.

We have investigated the involvement of specific phospholipase systems and their possible mutual relationship with the mechanism by which atrial natriuretic factor (ANF) increases phosphatidate (PA) and diacylglycerol (DAG) in rat aortic smooth muscle cells (RASMC), one of the major targets of this hormone. Our results indicate that ANF initially stimulates a phosphatidylinositol-dependent phospholipase C (PI-PLC) with a significant increase of DAG, enriched in arachidonate, and inositol trisphosphate (IP3) and then a phosphatidylcholine-dependent phospholipase C (PC-PLC) with formation of DAG, enriched in myristate, and phosphocholine (Pcho). Moreover, ANF stimulates PA formation at an intermediate stage between early and late DAG formation.

Proliferative effect of ammodytin L from the venom of Vipera ammodytes on 208F rat fibroblasts in culture.

Ammodytin L, purified from the venom of Vipera ammodytes, triggers a rapid and dramatic lytic process in myotubes in vitro, as well as in differentiated muscle cells in vivo, through a mechanism that is not well understood. Despite its great sequence similarity to phospholipase A2, it is devoid of any enzyme activity. Data on artificial membranes demonstrating a direct interaction between this toxin and the hydrophobic core of the lipid bilayer suggest that the toxin also acts on the lipid microenvironment in cell membranes.

Effect of ammodytin L from the venom of Vipera ammodytes on Xenopus laevis differentiated muscle fibres and regenerating limbs.

Ammodytin L is a non-catalytic, phospholipase-like snake venom toxin from Vipera ammodytes, which shows a cytotoxic activity on differentiated myotubes when tested in vitro. In the range of concentrations in which ammodytin L induced necrosis of myogenic cells in culture, other cell types (erythrocytes, platelets, fibroblasts) did not appear to be affected. To test the in vivo toxicity and the effective cytolytic specificity of ammodytin L we have followed the morphological changes in muscle tissue of Xenopus laevis limbs after intramuscular toxin injection.

Effect of ammodytin L from Vipera ammodytes on L-6 cells from rat skeletal muscle.

Ammodytin L (AMDL) is a myotoxic phospholipase-like protein from the venom of Vipera ammodytes with a serine in position 49 instead of an aspartate, therefore this toxin is devoid of phospholipase activity, and the membrane-damaging effect does not involve any step of phospholipase activity. The aim of the present study was to analyze the effect of AMDL on L-6 cells from rat skeletal muscle to investigate its mechanism of action and the role of calcium ions in its muscle-damaging activity. Our data indicate that the effect of ammodytin L is strongly dependent on the degree of cell differentiation.