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The purpose of this study was to examine the free radical scavenging and antioxidant activities of ellagic acid (EA) and ellagic acid peracetate (EAPA) by measuring their reactions with the radicals, 2,2-diphenyl-1-picrylhydrazyl and galvinoxyl using EPR spectroscopy. We have also evaluated the influence of EA and EAPA on the ROS production in L-6 myoblasts and rat liver microsomal lipid peroxidation catalyzed by NADPH. The results obtained clearly indicated that EA has tremendous ability to scavenge free radicals, even at concentration of 1 µM. Interestingly even in the absence of esterase, EAPA, the acetylated product of EA, was also found to be a good scavenger but at a relatively slower rate. Kinetic studies revealed that both EA and EAPA have ability to scavenge free radicals at the concentrations of 1 µM over extended periods of time. In cellular systems, EA and EAPA were found to have similar potentials for the inhibition of ROS production in L-6 myoblasts and NADPH-dependent catalyzed microsomal lipid peroxidation.

Integrin αvβ3, a cell surface receptor, participates in signaling transduction pathways in cancer cell proliferation and metastasis. Several ligands bind to integrin αvβ3 to regulate proliferation and metastasis in cancer cells. Crosstalk between the integrin and other signal transduction pathways also plays an important role in modulating cancer proliferation. Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) activates the downstream integrin FAK to stimulate biological activities including cancer proliferation and metastasis. Blockage of signals related to integrin αvβ3 was shown to be a promising target for cancer therapies. 3,3′,5,5′-tetraiodothyroacetic acid (tetrac) completely binds to the integrin with the thyroid hormone to suppress cancer proliferation. The (E)-stilbene analog, resveratrol, also binds to integrin αvβ3 to inhibit cancer growth. Recently, nanotechnologies have been used in the biomedical field for detection and therapeutic purposes. In the current review, we show and evaluate the potentiation of the nanomaterial carrier RGD peptide, derivatives of PLGA-tetrac (NDAT), and nanoresveratrol targeting integrin αvβ3 in cancer therapies.

Some constituents of the Mediterranean diet, such as extra-virgin olive oil (EVOO) contain substances such as hydroxytyrosol (HT) and its metabolite homovanillic alcohol (HA). HT has aroused much interest due to its antioxidant activity as a radical scavenger, whereas only a few studies have been made on the HA molecule. Both chemical synthesis and extraction techniques have been developed to obtain these molecules, with each method having its advantages and drawbacks. In this study, we report the use of tyrosol from olive mill wastewaters as a starting molecule to synthesize HT and HA, using a sustainable procedure characterized by high efficiency and low cost. The effects of HT and HA were evaluated on two cell lines, THP-1 human leukemic monocytes and L-6 myoblasts from rat skeletal muscle, after treating the cells with a radical generator. Both HT and HA efficiently inhibited ROS production. In particular, HT inhibited the proliferation of the THP-1 leukemic monocytes, while HA protected L-6 myoblasts from cytotoxicity.

Cancer resistance to chemotherapeutic agents is a major issue in the management of cancer patients. Overexpression of the ribonucleotide reductase regulatory subunit M2 (RRM2) has been associated with aggressive cancer behavior and chemoresistance. Nano-diamino-tetrac (NDAT) is a nanoparticulate derivative of tetraiodothyroacetic acid (tetrac), which exerts anticancer properties via several mechanisms and downregulates RRM2 gene expression in cancer cells. Resveratrol is a stilbenoid phytoalexin which binds to a specific site on the cell surface integrin αvβ3 to trigger cancer cell death via nuclear translocation of COX-2. Here we report that resveratrol paradoxically activates RRM2 gene expression and protein translation in colon cancer cells. This unanticipated effect inhibits resveratrol-induced COX-2 nuclear accumulation. RRM2 downregulation, whether achieved by RNA interference or treatment with NDAT, enhanced resveratrol-induced COX-2 gene expression and nuclear uptake which is essential to integrin αvβ3-mediated-resveratrol-induced antiproliferation in cancer cells. Elsewhere, NDAT downregulated resveratrol-induced RRM2 expression in vivo but potentiated the anticancer effect of the stilbene. These findings suggest that RRM2 appears as a cancer cell defense mechanism which can hinder the anticancer effect of the stilbene via the integrin αvβ3 axis. Furthermore, the antagonistic effect of RRM2 against resveratrol is counteracted by the administration of NDAT.

The molecular pathogenesis of colorectal cancer encompasses the activation of several oncogenic signaling pathways that include the Wnt/β-catenin pathway and the overexpression of high mobility group protein A2 (HMGA2). Resveratrol – the polyphenolic phytoalexin – binds to integrin αvβ3 to induce apoptosis in cancer cellsviacyclooxygenase 2 (COX-2) nuclear accumulation and p53-dependent apoptosis. Tetraiodothyroacetic acid (tetrac) is a de-aminated derivative ofl-thyroxine (T4), which – in contrast to the parental hormone – impairs cancer cell proliferation. In the current study, we found that tetrac promoted resveratrol-induced anti-proliferation in colon cancer cell lines, in primary cultures of colon cancer cells, andin vivo. The mechanisms implicated in this action involved the downregulation of nuclear β-catenin and HMGA2, which are capable of compromising resveratrol-induced COX-2 nuclear translocation. Silencing of either β-catenin or HMGA2 promoted resveratrol-induced anti-proliferation and COX-2 nuclear accumulation which is essential for integrin αvβ3-mediated-resveratrol-induced apoptosis in cancer cells. Concurrently, tetrac enhanced nuclear abundance of chibby family member 1, the nuclear β-catenin antagonist, which may further compromise the nuclear β-catenin-dependent gene expression and proliferation. Taken together, these results suggest that tetrac targets β-catenin and HMGA2 to promote resveratrol-induced-anti-proliferation in colon cancers, highlighting its potential in anti-cancer combination therapy.

In line with our previous studies, we have developed through a rational design approach novel morpholine and benzoxa(or thia)zine lead compounds that modulate a multiplicity of targets against atherosclerosis. We have evaluated the most promising compounds for their efficiency to a) interact and scavenge free radicals, b) inhibit the metal ion (Cu2+)-induced LDL oxidation c) act intracellularly as antioxidants in THP-1 monocytes from a leukemic patient and d) inhibit the pro-inflammatory enzymes cycloxygenase- 1 (COX-1) and -2 (COX-2) in vitro. Furthermore, two representative compounds were tested for their potential to decrease lipidemic parameters (TC, LDL and TG) in hyperlipidemic mice. Most derivatives indicated a remarkable antioxidant activity, while at the same time exhibited a significant in vitro anti-inflammatory activity, inhibiting COX-1 or/and COX-2 activity at 20 μΜ. Furthermore, after their long-term administration, compounds 6 and 8 afforded considerable activity in a chronic experimental animal model of hyperlipidemia (after high fat diet administration). The multifunctional pharmacological profile exhibited by the compounds of this study renders them interesting lead compounds for the development of novel agents against atherosclerosis.

In order to better understand the antioxidant behavior of a series of polyphenolic 2'-hydroxychalcones, we describe the results of several chemical and biological studies, in vitro and in vivo. Single crystal X-ray methods elucidated their molecular structures and important intermolecular interactions such as H-bonding and molecular stacking in the crystal structures that contribute to our knowledge in explaining antioxidant activity. The results of experiments using the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) UV-vis spectroscopic method indicate that a hydroxyl group in position 5' induces the highest antioxidant activity. Consequently, 2,2',5'-trihydroxychalcone was selected for further study in vitro towards ROS scavenging in L-6 myoblasts and THP-1 human monocytes, where it shows an excellent antioxidant activity in a concentration range lower than that reported by most studies of related molecules. In addition, this chalcone shows a very selective activity: it inhibits the proliferation of leukemic cells, but it does not affect the normal L-6 myoblasts and human fibroblasts. In studying 2,2',5'-trihydroxychalcone's effect on weight gain and serum glucose and insulin levels in Zucker fatty (fa(-)/fa(-)) rats we found that supplementing the diet with a 10 mg/kg dose of this chalcone (3 times weekly) blunted the increase in glucose that co-occurs with weight gain over the 6-week treatment period. It is concluded that 2,2',5'-trihydroxychalcone has the potential to serve as a protective agent for some debilitating diseases.

We report the results of in vivo studies in Caenorhabditis elegans nematodes in which addition of extra virgin olive oil (EVOO) to their diet significantly increased their life span with respect to the control group. Furthermore, when nematodes were exposed to the pesticide paraquat, they started to die after two days, but after the addition of EVOO to their diet, both survival percentage and lifespans of paraquat-exposed nematodes increased. Since paraquat is associated with superoxide radical production, a test for scavenging this radical was performed using cyclovoltammetry and the EVOO efficiently scavenged the superoxide. Thus, a linear correlation (y = -0.0838x +19.73, regression factor = 0.99348) was observed for superoxide presence (y) in the voltaic cell as a function of aliquot (x) additions of EVOO, 10 μL each. The originally generated supoeroxide was approximately halved after 10 aliquots (100 μL total). The superoxide scavenging ability was analyzed, theoretically, using Density Functional Theory for tyrosol and hydroxytyrosol, two components of EVOO and was also confirmed experimentally for the galvinoxyl radical, using Electron Paramagnetic Resonance (EPR) spectroscopy. The galvinoxyl signal disappeared after adding 1 μL of EVOO to the EPR cell in 10 minutes. In addition, EVOO significantly decreased the proliferation of human leukemic THP-1 cells, while it kept the proliferation at about normal levels in rat L6 myoblasts, a non-tumoral skeletal muscle cell line. The protection due to EVOO was also assessed in L6 cells and THP-1 exposed to the radical generator cumene hydroperoxide, in which cell viability was reduced. Also in this case the oxidative stress was ameliorated by EVOO, in line with results obtained with tetrazolium dye reduction assays, cell cycle analysis and reactive oxygen species measurements. We ascribe these beneficial effects to EVOO antioxidant properties and our results are in agreement with a clear health benefit of EVOO use in the Mediterranean diet.

Developing a rational strategy to control intracellular reactive oxygen species (ROS) requires understanding the mechanism of antioxidant activity. In this investigation the properties of a novel synthetic analog of vitamin E (IRFI005) with potent antioxidant activity are described. A mechanism is proposed for its efficient radical-scavenging effects. Cellular antioxidant and antitoxicity assays showed IRFI005 to freely permeate across cellular membranes, enabling it to be an effective suppressor of intracellular ROS and to protect cells against toxicity induced by free radical generating compounds. The free radical-scavenging activity of IRFI005 examined by UV-Vis and electron spin resonance (ESR) techniques clearly confirmed a "two electrons and/or H-atom" donation mechanism for each molecule of IRFI005. Reducing power assay as well as semi-empirical calculations revealed that under physiological conditions (pH∼7) almost all IRFI005 molecules are in the anionic state (IRFI005(-)). Data indicated that the electron donating ability of IRFI005(-) was dominant at physiological pH because of higher stability of quinine-IRFI005(-) and less barrier energy of IRFI005(-) than neutral IRFI005. Consequently, the efficient cellular protection of IRFI005 against toxic free radicals can be explained by a two electron-transfer process, because of reduced inter-frontier molecular orbital energy gap barrier at physiological pH. Our findings suggest that hydrophilic vitamin E-like antioxidants are good candidates in designing novel therapeutic strategies for inhibition of oxidative stress associated with different human diseases.

Nongenomic effects of thyroid hormones on Na+-K+-ATPase activity were studied in chick embryo hepatocytes at two different developmental stages, 14 and 19 days of embryonal age, and the signal transduction pathways involved were characterized. Our data showed the following. 1) 3,5,3′-Triiodo-l-thyronine (T3) and 3,5-diiodo-l-thyronine (3,5-T2) rapidly induced a transient inhibitory effect on the Na+-K+-ATPase; the extent and duration depended on the developmental age of the cells. 2) 3,5-T2behaved as a true hormone and fully mimicked the effect of T3. 3) Thyroxine had no effect at any of the developmental stages. 4) The inhibition of Na+-K+-ATPase was mediated by activation of protein kinase A, protein kinase C, and phosphoinositide 3-kinase, suggesting several modes of modulation of ATPase activity through phosphorylation at different sites. 5) The MAPK pathway did not seem to be involved in the early phase of hormone treatment. 6) The nonpermeant analog T3-agarose inhibited Na+-K+-ATPase activity in the same way as T3, confirming that hormone signaling initiated at a receptor on the plasma membrane. From these results, it can be concluded that the cell response mechanisms change rapidly and drastically within the early phase of embryo growth. The differences found at the two stages probably reflect the different roles of thyroid hormones during development and differentiation.

L-valine is a branched-chain amino acid (BCAA) largely used as dietary integrator by athletes and involved in some inherited rare diseases such as maple syrup urine disease. This pathology is caused by an altered BCAA metabolism with the accumulation of toxic keto acids in tissues and body fluids with consequent severe neurological symptoms. In animal models of BCAA accumulation, increased oxidative stress levels and lipid peroxidation have been reported. The aim of this study was to analyze both whether high BCAA concentrations in neurons induce reactive oxygen species (ROS) production and whether, by performing electrophysiological recordings, the neuronal functional properties are modified. Our results demonstrate that in primary cortical cultures, a high dose of valine increases ROS production and provokes neuronal hyperexcitability because the action potential frequencies and the persistent sodium current amplitudes increase significantly compared to non-treated neurons. Since Baicalein, a flavone obtained from the Scutellaria root, has been shown to act as a strong antioxidant with neuroprotective effects, we evaluated its possible antioxidant activity in primary cortical neurons chronically exposed to L-valine. The preincubation of cortical neurons with Baicalein prevents the ROS production and is able to revert both the neuronal hyperexcitability and the increase of the persistent sodium current, indicating a direct correlation between the ROS production and the altered physiological parameters. In conclusion, our data show that the electrophysiological alterations of cortical neurons elicited by high valine concentration are due to the increase in ROS production, suggesting much caution in the intake of BCAA dietary integrators.

Polyphenolic coumarins are known to act as antioxidants in biological systems, but it is difficult to distinguish their antioxidant activity from the many other effects they produce in cells. We have determined the radical scavenging capacity of 22 structurally related natural and synthetic 4-methylcoumarins, by measuring their reaction with radicals, galvinoxyl and 2,2-diphenyl-1-picrylhydrazyl, using electron paramagnetic resonance spectroscopy. Efficient antioxidant activity of 4-methylcoumarins in cells was verified using the DCF fluorescent probe assay for determination of intracellular reactive oxygen species levels. As expected, the o-dihydroxysubstituted coumarins were found to be excellent radical scavengers and better than the m-dihydroxysubstituted or monohydroxysubstituted analogues, but surprisingly the corresponding o-diacetoxy derivatives also turned out to be good scavengers, even in the absence of an esterase. Another unexpected result was that the anti-oxidant efficiency of 4-methylcoumarins could be modulated by introducing an ethoxycarbonyl-ethyl substituent at the C-3 position; this effect cannot be explained by simple electron donating/withdrawing properties. Coumarin concentrations of 10 μm or less were used in all experiments, corresponding to the levels relevant for therapeutic purposes. Considering that 4-methylcoumarins, in contrast to many other coumarins, are not metabolized to toxic epoxide intermediates, these results indicate promising new strategies for the design of non-toxic antioxidant coumarin-based drugs.

Non-Alcoholic Fatty Liver Disease (NAFLD) is nowadays considered one of the most diffused pathologic conditions affecting the liver especially in the Western world. The pathological mechanisms that induce the development of NAFLD are so far not completely clarified, even if now most researchers believe that an important condition that has to be satisfied is a significant accumulation of lipids inside the hepatocyte, principally as triglycerides. In the liver, such a lipid accumulation leads to an impairment of insulin signaling, which typically results in a failed phosphorylation of Akt, inducing the onset of insulin resistance. In the last years many researchers and clinicians focused their attention on the role played by fructose in metabolic diseases, such as obesity, type 2 diabetes, metabolic syndrome, as well as in liver specific pathologies, such as NAFLD. This huge debate about the role of fructose probably rose as a consequence of the recent increased diffusion of sweetened foods and beverages. In this context, the aim of this work was to develop an in vitro model for NAFLD, with particular reference to the role played by oleic acid and fructose in the onset of steatosis and insulin resistance. Moreover, we tested the effects of 3,5-diiodothyronine on steatosis and insulin resistance, trying to assess the potential mechanisms of action involved, considering the increasing interest about its role as a biologically active molecule. Our results show that, differently to oleic acid, fructose per se, in the range of physiological concentrations, is not able to induce neither triglycerides accumulation nor insulin resistance. Moreover we demonstrated that 3,5-diiodothyronine, at physiological levels, reduces triglycerides content and stimulates phosphorylation of Akt.

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 T4and IGF-I in rat L6 myoblasts, we have examined integrin αvβ3-mediated modulatory actions of T4on 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. IGF-I stimulated glucose transport and cell proliferation via the cell surface IGF-I receptor (IGFIR) and, downstream of the receptor, by the phosphatidylinositol 3-kinase signal transduction pathway. Addition of 0.1 nM free T4caused little or no cell proliferation but prevented both glucose uptake and proliferative actions of IGF-I. These actions of T4were mediated by an Arg-Gly-Asp (RGD)-sensitive pathway, suggesting the existence of crosstalk between IGFIR and the T4receptor located near the RGD recognition site on the integrin. An RGD-sequence-containing integrin inhibitor, a monoclonal antibody to αvβ3, and the T4metabolite tetraiodothyroacetic acid all blocked the inhibition by T4of IGF-I-stimulated glucose uptake and cell proliferation. Western blotting confirmed roles for activated phosphatidylinositol 3-kinase and extracellular regulated kinase 1/2 (ERK1/2) in the effects of IGF-I and also showed a role for ERK1/2 in the actions of T4that modified the effects of IGF-I. We conclude that thyroid hormone inhibits IGF-I-stimulated glucose uptake and cell proliferation in L6 myoblasts.

P-glycoprotein (P-gp; multidrug resistance pump 1, MDR1; ABCB1) is a plasma membrane efflux pump that when activated in cancer cells exports chemotherapeutic agents. Transcription of the P-gp gene (MDR1) and activity of the P-gp protein are known to be affected by thyroid hormone. A cell surface receptor for thyroid hormone on integrinαvβ3 also binds tetraiodothyroacetic acid (tetrac), a derivative of L-thyroxine (T4) that blocks nongenomic actions of T4and of 3,5,3′-triiodo-L-thyronine (T3) atαvβ3. Covalently bound to a nanoparticle, tetrac as nanotetrac acts at the integrin to increase intracellular residence time of chemotherapeutic agents such as doxorubicin and etoposide that are substrates of P-gp. This action chemosensitizes cancer cells. In this review, we examine possible molecular mechanisms for the inhibitory effect of nanotetrac on P-gp activity. Mechanisms for consideration include cancer cell acidification via action of tetrac/nanotetrac on the Na+/H+exchanger (NHE1) and hormone analogue effects on calmodulin-dependent processes and on interactions of P-gp with epidermal growth factor (EGF) and osteopontin (OPN), apparently viaαvβ3. Intracellular acidification and decreased H+efflux induced by tetrac/nanotetrac via NHE1 is the most attractive explanation for the actions on P-gp and consequent increase in cancer cell retention of chemotherapeutic agent-ligands of MDR1 protein.

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 mu M cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.

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. Low doses of myotoxin gave rise to a marked release of creatine kinase in myotubes differentiated from L-6 myoblasts and the presence of calcium ions plays a role in the cytotoxic effect. The presence of EGTA in the incubation buffer reduced by 50% the release of creatine kinase. No membrane damage was observed in myoblasts, but there was a significant increase of intracellular calcium concentration measured with Fura-2. A non-specific membrane effect of AMDL was ruled out using platelets as reference cells: no platelet aggregation pattern and no increase in intracellular calcium were observed.

The sodium/proton (Na/H) exchanger, Na,K-ATPase, and Ca2+-ATPase are membrane ion pumps whose basal activities may be regulated by local nongenomic actions of thyroid hormone and hormone analogues via a hormone receptor on plasma membrane integrin αvβ3. System A amino acid transport and the activity of P-glycoprotein (P-gp; ABCB1), a multidrug efflux pump, are also modulated by thyroid hormone and αvβ3. Where signal transduction has been studied, the presence of the hormone at the receptor is transduced by mitogen-activated protein kinase (MAPK) isoforms (ERK1/2; p38) or phosphatidylinositol 3-kinase into local actions. The existence of the cell surface receptor offers opportunities to pharmacologically modify actions of these important transport functions with nanoparticulate formulations of T4 and T3 that do not enter the cell. Such formulations may reverse complex intracellular accumulations of H+, Na+, and Ca2+ that occur in clinical settings such as ischemia. In addition, nanoparticulate tetraiodothyroacetic acid (tetrac), a thyroid hormone analogue that inhibits binding of T4 and T3 to integrin αvβ3 as well as certain other functions of the integrin, may reverse P-gp-dependent resistance to anti-cancer drugs in tumor cells.

Administration of chemotherapy during pregnancy may represent a big risk factor for the developing brain, therefore we studied whether the transplacental transport of doxorubicin (DOX) may affect the development of neuroendocrine system. DOX (25 mg/kg; 3 times interaperitoneally/week) was given to pregnant rats during whole gestation period. The disturbances in neuroendocrine functions were investigated at gestation day (GD) 15 and 20 by following the maternal and fetal thyroid hormone levels, fetal nucleotides (ATP, ADP, AMP) levels and adenosine triphosphatase (Na(+), K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase) activities in two brain regions, cerebrum and cerebellum. In control group, the levels of maternal and fetal serum thyroxine (T4), triiodothyronine (T3), thyrotropin (TSH), and fetal serum growth hormone (GH) increased from days 15 to 20, whereas in the DOX group, a decrease in maternal and fetal T4, T3 and increase in TSH levels (hypothyroid status) were observed. Also, the levels of fetal GH decreased continuously from GD 15 to 20 with respect to control group. In cerebrum and cerebellum, the levels of fetal nucleotides and the activities of fetal ATPases in control group followed a synchronized course of development. The fetal hypothyroidism due to maternal administration of DOX decreased the levels of nucleotides, ATPases activities, and total adenylate, instead, the adenylate energy charge showed a trend to an increase in both brain regions at all ages tested. These alterations were dose- and age-dependent and this, in turn, may impair the nerve transmission. Finally, DOX may act as neuroendocrine disruptor causing hypothyroidism and fetal brain energetic dysfunction.

Thyroid hormones (THs) have a wide variety of essential roles in vertebrates, ranging from the regulation of key metabolic processes to cell proliferation and apoptosis. The classical mechanism of action of THs is genomic; 3,5,3'-triiodothyronine (T3) binds to specific nuclear receptors (TRs) and modifies the expression of specific genes. Recently, a new category of mechanisms, termed nongenomic, has been discovered for T3. These mechanisms include, among others, the rapid activation of signal transduction pathways, such as PI3K/Akt and MAPK, which eventually lead to cell proliferation. These effects are mediated in some cell types by a plasma membrane receptor, identified as integrin αvβ3, and in other cell types by cytoplasmic TRβ1. The aim of this work was to analyze the effect of T3 on the cell growth of chick embryo hepatocytes at two different stages of development, 14 and 19 days, and to determine the activation of the signal transduction pathways, focusing on the potential involvement of a plasma membrane receptor and the possible participation of PI3K/Akt and reactive oxygen species (ROS). Our results clearly show that T3 stimulates cell proliferation at both stages of development through the activation of the PI3K/Akt pathway and the production of small amounts of ROS, which operate as effective second messengers. Moreover, we prove that these effects are not initiated at the plasma membrane receptor for T3.

The insulin modulation of Na-H antiport in rat hepatocytes was studied using the fluorescent, pH-sensitive intracellular probe, 2',7' bis (carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Our data show that insulin stimulates the Na-H antiport. The dose-response of insulin effect shows a behavior typical of other insulin responses: a maximum in the physiological range (1 nM) and smaller effects at higher and lower hormone concentrations. The time-course of activation is very fast at high hormone concentrations and slow, but reaching a higher value, for the physiological concentrations (0.26 +/- 0.05 and 0.18 +/- 0.022 pH units for 1 nM and 1 microM insulin respectively). The use of phorbol, 12-myristate, 13-acetate (PMA), a potent activator of protein kinase C and its inhibitor staurosporine, and the inhibitor of tyrosine kinase erbstatin analog, suggests that both protein kinase C and tyrosine kinase could be involved in the mechanism leading to Na-H antiport activation by insulin. We suggest that the activation of the antiport involves the two pathways depending on the hormone concentration. In particular, protein kinase C would mediate the effects of high hormone concentrations, acting as a growth factor, since staurosporine fully inhibited insulin 1 microM, but only partially 1 nM effects, and tyrosine kinase would mediate the effect of insulin 1 nM and only partially 1 microM. Okadaic acid 1 microM, a potent inhibitor of protein phosphatases, mimicked the hormone effects on the antiport and abolished the different time-course due to hormone concentration, suggesting a role of kinases and phosphatases in the signal transduction. The effect of all activators was abolished by amiloride analog, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), confirming the specificity of these effects.

""It is now widely accepted that thyroid hormones, L-thyroxine (T4) and 3,30,5-triiodo-L-thyronine (T3), 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 T3 or T4 and effects on the immune responses. Integrins are extracellular matrix proteins that are. important modulators of many cellular responses, and the integrin avb3 has been identified as a cell surface. receptor for thyroid hormones. Rapid signaling via this plasma membrane binding site appears to be. responsible for many nongenomic effects of thyroid hormones, independent of the classic nuclear receptors.. Through the integrin avb3 receptor the hormone can activate both the ERK1\\\/2 and phosphatidylinositol. 3-kinase pathways, with downstream effects including intracellular protein trafficking,. angiogenesis and tumor cell proliferation. It has recently become clear that an important downstream. target of the thyroid hormone nongenomic pathway may be the mammalian target of rapamycin, mTOR.. New results demonstrate the capability of T3 or T4 to induce in the short time range important responses. related to the immune function, such as reactive oxygen species production and cell migration in THP-1. monocytes. Thus thyroid hormones seem to be able to modulate the immune system by a combination of. rapid nongenomic responses interacting with the classical nuclear response.. ""

A large series of hydroxytyrosyl esters of C2-C18 fatty acids with increasing lipophilicity was prepared by a new highly efficient method based on acylation of methylorthoformate-protected hydroxytyrosol. All products were tested for the relative antioxidant effect using ABTS assays in ethanolic medium and DCF assays in L6 cells. No linear correlation between lipophilicity and antioxidant effect was found. ABTS assays showed a growing antioxidant capacity, with respect to hydroxytyrosol, only for medium-sized ester chains (C4-C10), and a nearly constant capacity for the higher homologues. This has been rationalized by molecular dynamics experiments in terms of partial shielding of the catecholic hydroxyls by long chained esters. A similar and dose-dependent pattern was observed in DCF assays in L6 cells, but a sharp antioxidant activity drop resulted for long chain esters, probably due to membrane entrapment.

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). Signal transduction from the alphaVbeta3 integrin may proceed through at least three independent pathways (protein kinase C, Src or mitogen-activated kinases) but the details are still unknown. Thyroid hormones induce nongenomic effects on at least three important Na(+)-dependent transport systems, the Na(+)/K(+)-ATPase, the Na(+)/H(+) exchanger, and amino acid transport System A, leading to a mitogenic response in embryo cells; but modulation of the same transport systems may have different roles in other cells and at different developmental stages. It seems that thyroid hormones in many cases can modulate nongenomically the same targets affected by the nuclear receptors through long-term mechanisms. Recent results on nongenomic effects confirm the old theory that the primary role of thyroid hormones is to keep the steady-state level of functioning of the cell, but more and more mechanisms are discovered by which this goal can be achieved.

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). In vascular cells and cardiomyocytes ANP stimulates the antioxidant defense, but in other systems such as hepatoblastoma and macrophages ANP may produce either antioxidant or prooxidant effects, depending on experimental conditions and cell context. At present very little is known on the relationship between ANP and ROS production in the normal homeostatic processes or during the development of cardiovascular diseases and cancer. Our current knowledge of the role of ANP in signaling pathways leading to the generation of intracellular messengers such as diacylglycerol (DAG), and guanosine 3'-5'-cyclic monophosphate has been examined in order to clarify the mechanisms by which the hormone may counteract or contribute to the potentially dangerous effects of free radicals.

Reactive oxygen species (ROS) is the common name used to describe the partially reduced forms of molecular oxygen that may be generated in cells during oxidative metabolism. They are normally considered to be toxic, and cells possess various defence systems to protect themselves including antioxidant enzymes and low molecular weight antioxidants like vitamin C and vitamin E. However, it is now clear that small amounts of ROS also act as messenger molecules in cell signal transduction pathways; the plasma membrane of eukaryotic cells in particular contains a variety of different ROS-producing oxidases and reductases, of which the best characterized are the superoxide-producing NADPH oxidases. It has been known for many years that membrane redox activity can be changed rapidly by various hormones and growth factors, but the molecular mechanisms involved and the physiological importance of this phenomenon have only recently begun to be unveiled. This review summarizes the state of the art on plasma membrane-based ROS signalling in the pathways of insulin, steroid and thyroid hormones and growth factors. The apparent paradox of ROS being essential biomolecules in the regulation of cellular functions, but also toxic by-products of metabolism, may be important for the pharmacological application of natural and synthetic antioxidants.

Extranuclear or nongenomic effects of thyroid hormones do not require interaction with the nuclear receptor, but are probably mediated by specific membrane receptors. This review will focus on the extranuclear effects of thyroid hormones on plasma membrane transport systems in non mammalian cells: chick embryo hepatocytes at two different stages of development, 14 and 19 days. At variance with mammals, the chick embryo develops in a closed compartment, beyond the influence of maternal endocrine factors. Thyroid hormones inhibit the Na+/K+-ATPase but stimulate the Na+/H+ exchanger and amino acid transport System A with different dose-responses: a bell-shaped curve in the case of the exchanger and a classic saturation curve in the case of System A. These effects are mimicked by the analog 3,5-diiodothyronine. Signal transduction is mediated by inter-play among kinases, mainly protein kinase C and the MAPK pathway, initially primed by second messengers such as Ca2+, IP3, and DAG as in mammalian cells. Thyroid hormones and 3,5-diiodothyronine stimulate thymidine incorporation and DNA synthesis, associated with the increased levels and activity of cyclins and cyclin-dependent kinases involved in the G1/S transition, and also these effects have their starting point at the plasma membrane. Increasing evidence now demonstrates that thyroid hormones act as growth factors for chick embryo hepatocytes and their extranuclear effects are important for prenatal development and differentiation. (c) 2005 Elsevier Inc.

The microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) is the rate-limiting enzyme in cholesterol synthesis and is highly regulated by a variety of factors.We have recently reported increased reductase activity during ageing, attributed to a higher activation state and impaired degradation of the hepatic enzyme. One of the widely recognized causes of age-related metabolic modifications is the large increase of reactive oxygen species (ROS). Therefore, the effect of ROS increase on the activity and the regulation of the HMGCoAR has been investigated in two different experimental models of ROS enriched tissue: liver from rats fed on diets deprived of either Vitamin E (Vit. E) or polyunsaturated fatty acids (Pufa). The results show that in these models, compared to that of old rats, full activation the HMGCoAR was detected while a different degradation rate is observed with the respect to old rats. Thus, our data show full correlation between ROS production and increased HMGCoAR activity. The possible therapeutic implications of these results are discussed. # 2005 Elsevier Ireland Ltd. All rights reserved.

L-T3 and L-T4 activated the Na+/H + exchanger of L-6 myoblasts, with a fast nongenomic mechanism, both in the steady state and when cells undergo acid loading with ammonium chloride. Monitored with the intracellular pH-sensitive fluorescent probe 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein, activation of the exchanger appeared to be initiated at the plasma membrane, because T 3-agarose reproduced the effect of L-T3, and triiodothyroacetic acid, a hormone analog previously shown to inhibit membrane actions of thyroid hormone, blocked the action of L-T3 on the exchanger. We show here for the first time that transduction of the hormone signal in this nongenomic response requires tyrosine kinase-dependent phospholipase C activation and two different signaling pathways: 1) mobilization of intracellular calcium, assessed by the fluorescent probe fura-2, through activation, of inositol tris-phosphate receptors and without contributions from extracellular calcium or ryanodine receptors; and 2) protein phosphorylation involving protein kinase C and MAPK (ERK1/2), as shown by the use of kinase inhibitors and by immunoblotting for activated kinases. Fil: D'Arezzo, Silvia. Università di Roma; Italia Fil: Incerpi, Sandra. Università di Roma; Italia Fil: Davis, Faith B.. Ordway Research Institute; Estados Unidos Fil: Acconcia, Filippo. Università di Roma; Italia Fil: Marino, Maria. Università di Roma; Italia Fil: Farias, Ricardo Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Davis, Paul J.. Ordway Research Institute; Estados Unidos

Low physiological concentrations of 17beta-estradiol increased the intracellular pH of rat aortic smooth muscle cells by a rapid nongenomic mechanism. This effect was due to stimulation of the Na+/H+ exchanger activity, measured using the intracellular pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. The 17beta-estradiol gave rise to a bell-shaped dose response, with a maximum at 10-12 m and no significant effect at 10-9 m. The specificity of the effect was verified by the use of the Na+/H+ exchanger inhibitor 5-(ethyl-N-isopropyl)amiloride and the lack of effect of the isomer 17alpha-estradiol. Inhibitors of the nuclear estrogen receptors, tamoxifen and ICI 182,780, completely prevented activation of the exchanger by 17beta-estradiol. The effect of low estrogen concentrations on the intracellular pH was mimicked by both norepinephrine and phenylephrine, suggesting a connection between the increase of intracellular pH and the muscle contraction process. The transduction mechanism for this nongenomic effect of estrogens did not involve modulation of the cAMP content, whereas inositol 1,4,5-trisphosphate, protein kinase C and MAPK pathways appear to play a role, as indicated by both pharmacological approaches and immunoblot experiments on protein kinase C translocation and ERK phosphorylation. These results for the first time provide evidence for a nongenomic effect of low physiological concentrations of 17beta-estradiol on intracellular pH that, together with other factors, may contribute to the development of hypertension and atherosclerosis in men and postmenopausal women and increase the risk of cardiovascular disease. Paradoxically, the lack of stimulation at high physiological estradiol levels could explain the protective effects found in premenopausal women.

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. The specificity of the effect was verified by the use of inhibitors and analogues. The thyroid hormone analog 3,5-diiodo-L-thyronine was able to mimic some of the hormone effects, but with a lower efficiency. The effect on the Na(+)/H(+) exchanger was identified for 14-d-old and 19-d-old cells, whereas the amino acid transport could only be activated at the late stage of embryo development. Both transport systems were activated through a signal transduction pathway involving PKC, MAPK pathway, and PI3K, even though the differences in response behavior indicate a differential modulation of the two transport systems by L-T(3) and L-T(4). These results clearly demonstrate the existence of rapid nongenomic action of thyroid hormones also in avian cells, and show that activation of System A amino acid transport is not directly correlated to changes in intracellular pH. For the first time, evidence is presented which suggests that short-term effects of thyroid hormones may play a role during fetal development and cell differentiation.

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. Hormone concentrations of 10(-10) M activate the antiport, increasing both the rate of recovery and the set point by approximately 0.2 pH units. This effect is mediated by diacylglycerol as a result of phospholipid hydrolysis by a phospholipase C, even if an involvement of adenosine 3',5'-cyclic monophosphate (cAMP) cannot be ruled out. ANF (10(-7) M) inhibits the antiport, decreasing both the rate of recovery and the set point by approximately 0.3 pH units, because of guanosine 3',5'-cyclic monophosphate production. Both inhibition and stimulation of pHi by ANF were more pronounced when the hormone was given before the acid load, perhaps because of the longer time exposure. We present new hypotheses on the mechanism of action of this paracrine/autocrine factor.

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 (ΔpH of 0.132 ± 0.007 and 0.160 ± 0.040 over basal value, for myoblasts and myotubes, respectively; means ± SD, n = 6–8) and smaller effects at higher and lower concentrations. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated the antiport in myoblasts but not in myotubes. Surprisingly the rapid increase in intracellular pH was not observed when insulin and PMA were added simultaneously to myoblasts; apparently these two activators mutually excluded each other. Downregulation of protein kinase C, obtained by preincubation of cells with PMA for 20 hr, totally abolished both hormone and PMA effects in myoblasts, whereas in myotubes insulin stimulation was not affected. Inhibitors of tyrosine kinase activity, such as erbstatin analog and genistein abolished insulin effect on the Na/H antiport, both in myoblasts and in myotubes. Different sensitivity to pertussis toxin in the two cell types suggests that the differentiation process leads to a change in the signal pathways involved in the physiological response to insulin. J. Cell. Physiol. 171:235–242, 1997. © 1997 Wiley-Liss, Inc.

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na+/H+ antiporter, the main system responsible for maintaining unaltered pHi in those cells. At the end of the infection period, a rise in both pHi value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.

We examined the effects of insulin on fatty acid uptake in L-cell fibroblasts, using cis -parinaric acid to measure uptake rates in the absence of esterification and [ 3 H]oleic acid to measure uptake rates in the presence of esterification. L-cells exhibited both high and low affinity insulin binding sites with K d of 23 n M and 220 n M and a cellular density of 1.4 and 6.8 × 10 5 sites/cell, respectively. Insulin in the range 10 −9 to 10 −7 M significantly decreased both the initial rate and maximal extent of cis -parinaric acid uptake by 24 to 30%. Insulin also reduced [ 3 H]oleic acid uptake up to 35%, depending on insulin concentration and decreased the amount of fatty acid esterified into the phospholipids and neutral lipids by 28 and 70%, respectively. In contrast, glucagon or epinephrine stimulated both the initial rate and extent of cis -parinaric acid uptake 18 and 25%, respectively. Because L-cells lack β-adrenergic receptors, the epinephrine effect was not the result of β-receptor stimulation. Hence, insulin altered not only fatty acid uptake, as determined by cis -parinaric and oleic acid uptake, but also altered the intracellular oleic acid esterification.

The modulation by epidermal growth factor (EGF) of the Na+/H+ antiport in fetal and adult rat hepatocytes was studied in nominally HCO3- free solution. EGF (10 nM) activated the antiport in adult rat hepatocytes by 0.22 +/- 0.03 (mean +/- SD;n=10) pH units over basal value, measured with the fluorescent pH-sensitive intracellular probe, 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein (BCECF). The effect of EGF was inhibited by amiloride analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA), by ouabain, inhibitor of the Na+ pump, and by erbstatin analogue, an inhibitor of the tyrosine kinase activity of the EGF receptor. The effect of EGF on Na+/H+ antiport in adult rat hepatocytes appeared to be mediated by both protein kinase C (PKC) and G protein system. No effect of EGF and phorbol 12-myristate 13-acetate, an activator of PKC, on the Na+/H+ antiport was observed in fetal hepatocytes of 20 and 22 days. A different sensitivity of the antiport to high concentrations of amiloride and EIPA suggests that altered amount of the Na+/H+ antiport units or different isoforms could be expressed in fetal compared with adult cells.

We have characterized a membrane-bound phosphatidylcholine (PC) specific phospholipase C (PC-PLC) in plasma membranes from rat cardiac muscle, and have investigated the role of PC-PLC and PC-specific phospholipase D (PC-PLD) activities in the mechanism of action of atrial natriuretic factor (ANF). In purified sarcolemma, ANF stimulated over a wide range of concentrations with a maximum at 10(-11) M the hydrolysis of phosphatidylcholine through PC-PLD giving phosphatidate and choline, whereas higher concentrations of ANF (10(-10) M) preferentially stimulated PC breakdown through PC-PLC to form diacylglycerol and phosphocholine. To confirm the involvement of the PC-PLD in the mechanism of ANF action, we measured the transphosphatidylation reaction, a specific assay for this phospholipase which in the presence of ethanol catalyses the phosphatidylethanol formation from PC. ANF stimulated phosphatidylethanol formation with the same dose-response behavior as phosphatidate formation. The significant diacylglycerol increase at 10(-10) M ANF, in the presence of propranolol, a potent inhibitor of phosphatidate phosphatase which can hydrolyse phosphatidate to give diacylglycerol, suggested a direct involvement of PC-PLC. The use of GTP-gamma-S, a non hydrolysable analog of GTP, and of pertussis toxin showed the involvement of a pertussis toxin insensitive G protein in PC-PLC mediated ANF signal transduction. We suggest a differential effect of ANF on PC breakdown by phospholipases C and D depending on the concentration of the peptide.

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose levels is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigated in vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accommodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtained in vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.

Although the intracellular fatty acid binding proteins have been investigated for nearly two decades and purified proteins are now available, little is known regarding the function of these proteins in intact cells. Therefore, L-cell fibroblasts transfected with cDNA encoding for rat liver fatty acid binding protein (L-FABP) were examined as to whether L-FABP expression in intact cells modifies plasma membrane enzyme activities, fluidity, and lipids. Plasma membrane Na/K-ATPase activity was 65.9 +/- 18.7 and 38.6 +/- 22.8 (P less than 0.001) nmol/mg protein x min for control and high-expression transfected cells, respectively. Consistent with this observation, [3H] ouabain binding to whole cells was significantly decreased from 3.7 +/- 0.3 to 2.0 +/- 0.8 pmol ouabain bound/mg cell protein in control and high-expression cells, respectively, whereas the cell's affinity for ouabain was not significantly altered. Unexpectedly, Western blot analysis indicated that transfected cells had higher levels of Na+, K(+)-ATPase protein; in contrast, the activities of 5'-nucleotidase and Mg-ATPase were unaltered. The effects of L-FABP expression on plasma membrane Na/K-ATPase function appeared to be mediated through alterations in plasma membrane lipids and/or structure. The plasma membrane cholesterol/phospholipid ratio decreased and the bulk plasma membrane fluidity increased in the high-expression cells. In conclusion, plasma membrane Na/K-ATPase activity in L cells may be regulated in part through expression of cytosolic L-FABP.

Insulin treatment of isolated liver plasma membranes induced the release of 5′-nucleotidase and alkaline phosphatase. This effect was maximal at physiological hormone concentrations, being 36% and 17% for 5′-nucleotidase and alkaline phosphatase respectively, and was fully mimicked by the phosphatidylinositol specific phospholipase C (PI-PLC), thus confirming the presence of a glycosyl-phosphatidylinositol anchoring-system for these exofacial enzymatic proteins. The complete inhibition of insulin dependent enzyme release by neomycin is strongly supportive of an involvement of membrane-located PI-PLC activity. In addition, the insulin-like effect on enzyme release induced by the GTP non-hydrolysable analog, GTP-γ-S, and its sensitivity to the pertussis toxin are in favour of a mediatory role exerted by the G proteins system, in the transduction of some actions of insulin.

Amphibia undergo regular annual cycles of metabolic activity that are influenced by both exogenous factors and hormones. Insulin binding to crude frog hepatic membranes was studied throughout the year. The general character of insulin binding was similar to that in other vertebrates; the maximum specific binding was achieved after 4 hr at 4 degrees, the optimum pH was 7.8, half-maximal displacement of bound insulin was from 9 x 10(-10) to 1 x 10(-9) M, and insulin analogs competed for the insulin receptor in line with their relative biological potencies. A biphasic Scatchard plot and negative cooperativity of the receptor were also observed in frog liver membranes. Affinity constants from Scatchard plots revealed high and low affinity binding sites which were unchanged during the year. The seasonal cycle, however, markedly affected the binding capacity for both sites. Maximum binding occurred in May-June and the minimum in November-December for both classes of receptors. Binding capacities ranged from 1.71 to 11.33 fmol/mg protein for the high affinity sites and from 432 to 3171 fmol/mg protein for the low affinity sites. It is concluded that annual cycles of insulin binding reflect modulation of receptor number rather than receptor affinity.

1. 1. The presence of a glucocorticoid soluble receptor is demonstrated in frog liver cytosol. 2. 2. The kinetic characterization of frog liver cytosolic receptor for glucocorticoids is reported and its steroid specificity assessed. 3. 3. Results indicate a gross similarity between frog liver and mammalian glucocorticoid receptor, being a major difference the reduced binding capacity.

1. 1. A circannual study of tyrosine aminotransferase and other metabolic enzymes in frog liver is reported. The subcellular distribution of all enzymatic activities under investigation was also studied. 2. 2. Results show significant oscillations of all enzymatic activities throughout the year; in particular tyrosine aminotransferase has a marked summer maximum. 3. 3. The subcellular distribution of tyrosine aminotransferase shows significant variations: the soluble activity of the enzyme presents a bimodal circannual distribution, which has its counterpart in an increased activity of heavier fractions.

Chlorpropamide and phenformin inhibited (Na+K+)-ATPase and stimulated a high affinity cyclic AMP-phosphodiesterase of isolated liver plasma membrane when tested in vitro. In addition, the two drugs decreased the intracellular cyclic AMP content of isolated hepatocytes without being effective on plasma membrane-bound adenylate cyclase. The results suggest that the plasma membrane plays an important role in the mechanism of action of the two hypoglycemic drugs, but do not exclude the presence of intracellular targets.

Insulin binding to isolated rat hepatocytes was studied during prenatal and postnatal life. Results show that in hepatocytes isolated from prenatal, postnatal and adult rat there is a constant increase in the number of insulin binding sites per cell, whereas the affinity of plasma membrane receptors for the hormonal ligand remains unaltered from prenatal to adult hepatocytes. Autoradiographic studies indicate a greater internalization of hormone during prenatal life and, taking into account the increase of cell size, suggest an unchanged surface density of receptor sites before and after birth.

Insulin receptors and glucose transport-inducing proteins have been extracted from rat liver membranes onto positively charged lipid bilayer vesicles. The extraction was carried out during the incubation of the vesicles with lipid vesicles caused an overall enhancement of specific insulin binding and of glucose transport inducement. The latter has been inferred from the oxidation rate of transported glucose through a spherical bilayer membrane entrapping the oxidizing glucose oxidase. Glucose transport is not enhanced by insulin binding, indicating that the two functions become dissociated when the proteins are transferred from the plasma membrane onto the bilayer vesicles.

1. 1. The presence of tyrosine aminotransferase is reported both in particulate and soluble fractions of frog liver. 2. 2. The activity of the soluble enzyme of frog liver was investigated with regard to its dose and time dependence, its substrate specificity and concentration dependence, its thermal sensitivity as well as pH and temperature dependence. 3. 3. It appears that the properties of the soluble tyrosine aminotransferase of frog liver are in close agreement with those reported for the mammalian liver enzyme.

The aim of the present study was to examine the possible physiological responsiveness of the sodium pump to insulin in rat muscle, an effect that has never been convincingly demonstrated. The insulin stimulation of the sodium pump was estimated by two well-established parameters: ouabain binding to pieces of soleus muscle, and Na/K-ATPase activity of purified plasma membranes. For both parameters the dose dependence of the insulin effect on the sodium pump shows the characteristic bell-shaped stimulation pattern, with a maximum in the physiological hormone concentration range. This result has not been observed in previous studies where insulin concentrations two to three orders of magnitude higher were used. It can be concluded that an effect of insulin on the regulation of the Na pump in muscle might well be operating in vivo.

Different features of insulin sensitivity have been considered, in rat hepatocytes, with respect to their age-dependence. In particular, plasma membrane-located responses such as (Na/K)-ATPase and Na-dependent aminoacid transport were studied together with cytoplasmic responses such as the insulin-stimulated tyrosine aminotransferase. It appears, as far as the insulin sensitivity is concerned, that the age of the animal does not affect plasma membrane-bound events whereas plasma membrane-mediated intracellular responses are definitely impaired. Insulin binding to intact hepatocytes does not seem to be age-dependent.

Colchicine effect has been tested on rat liver plasma membrane-bound enzymes after in vitro or in vivo treatment. It appears that the in vitro treatment does not affect 5'-nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase, whereas adenylate cyclase is sensitive to both in vitro and in vivo treatment, the latter condition being also effective for 5'-nucleotidase.