Short-Term Activation by Low 17β-Estradiol Concentrations of the Na+/H+ Exchanger in Rat Aortic Smooth Muscle Cells: Physiopathological Implications

Low physiological concentrations of 17β-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 17β-estradiol gave rise to a bell-shaped dose response, with a maximum at 10−12m and no significant effect at 10−9m. 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 17α-estradiol. Inhibitors of the nuclear estrogen receptors, tamoxifen and ICI 182,780, completely prevented activation of the exchanger by 17β-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 17β-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 3,5,3′-triiodo-L-thyronine on the intracellular pH of L-6 myoblasts are mediated by intracellular calcium mobilization and kinase pathways

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; ItaliaFil: Incerpi, Sandra. Università di Roma; ItaliaFil: Davis, Faith B.. Ordway Research Institute; Estados UnidosFil: Acconcia, Filippo. Università di Roma; ItaliaFil: Marino, Maria. Università di Roma; ItaliaFil: 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; ArgentinaFil: Davis, Paul J.. Ordway Research Institute; Estados Unido

Tetrac and NDAT Induce Anti-proliferation via Integrin αvβ3 in Colorectal Cancers With Different K-RAS Status

Colorectal cancer is a serious medical problem in Taiwan. New, effective therapeutic approaches are needed. The selection of promising anticancer drugs and the transition from pre-clinical investigations to clinical trials are often challenging. The deaminated thyroid hormone analog (tetraiodothyroacetic acid, tetrac) and its nanoparticulate analog (NDAT) have been shown to have anti-proliferative activity in vitro and in xenograft model of different neoplasms, including colorectal cancers. However, mechanisms involved in tetrac- and NDAT-induced anti-proliferation in colorectal cancers are incompletely understood. We have investigated possible mechanisms of tetrac and NDAT action in colorectal cancer cells, using a perfusion bellows cell culture system that allows efficient, large-scale screening for mechanisms of drug actions on tumor cells. Although integrin αvβ3 in K-RAS wild type colorectal cancer HT-29 cells was far less than that in K-RAS mutant HCT116 cells, HT-29 was more sensitive to both tetrac and NDAT. Results also indicate that both tetrac and NDAT bind to tumor cell surface integrin αvβ3, and the agents may have different mechanisms of anti-proliferation in colorectal cancer cells. K-RAS status appears to play an important role in drug resistance that may be encountered in treatment with this drug combination

NONGENOMIC EFFECTS OF THYROID HORMONES ON ION TRANSPORT

Extranuclear or nongenomic effects of thyroid hormone are unaffected by the inhibitors of protein synthesis, and their time course cannot be explained by the interaction of the hormone molecule with nuclear receptors. Their site of action has been localized at the plasma membrane and identified very recently. In particular thyroid hormone has been reported to activate, by both genomic and non genomic mechanism, the Ca2+-ATPase, an ion pump that removes calcium from the cytosol and stores it in the sarcoplasmic reticulum. The decrease in intracellular Ca2+ generated during the systole leads to cardiac muscle relaxation. Given to all these important effects on the cardiovascular system, T3 can be also envisaged as a potent novel therapeutic agent as a inotropic drug. The Na/H exchanger, activated in L-6 myoblasts by the thyroid hormone through both genomic and nongenomic mechanism, is a ubiquitous plasma membrane integral protein exchanging extracellular Na+ with cytoplasmic H+ ions according to the concentration gradient; it does not require energy supply, but depends on the Na+/K+-ATPase. The 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, through activation of IP3-receptors and without contributions from extracellular calcium or ryanodine receptors; 2) protein phosphorylation involving protein kinase C and mitogen-activated protein kinase (MAPK; ERK1/2), likely interfaces between genomic and nongenomic effects. The extranuclear actions should be taken into account when considering appropriate therapeutic intervention based on thyroid hormone both for their possible reinforcement of the nuclear responses and for their fast time onset, that might be of clinical relevance particularly for the cardiovascular system. Department of Biology, University of Rome “Roma Tre”, Viale Extranuclear or nongenomic effects of thyroid hormone are unaffected by the inhibitors of protein synthesis, and their time course cannot be explained by the interaction of the hormone molecule with nuclear receptors. Their site of action has been localized at the plasma membrane and identified very recently. In particular thyroid hormone has been reported to activate, by both genomic and non genomic mechanism, the Ca2+-ATPase, an ion pump that removes calcium from the cytosol and stores it in the sarcoplasmic reticulum. The decrease in intracellular Ca2+ generated during the systole leads to cardiac muscle relaxation. Given to all these important effects on the cardiovascular system, T3 can be also envisaged as a potent novel therapeutic agent as a inotropic drug. The Na/H exchanger, activated in L-6 myoblasts by the thyroid hormone through both genomic and nongenomic mechanism, is a ubiquitous plasma membrane integral protein exchanging extracellular Na+ with cytoplasmic H+ ions according to the concentration gradient; it does not require energy supply, but depends on the Na+/K+-ATPase. The 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, through activation of IP3-receptors and without contributions from extracellular calcium or ryanodine receptors; 2) protein phosphorylation involving protein kinase C and mitogen-activated protein kinase (MAPK; ERK1/2)

Nongenomic effects of thyroid hormones on ion transport

Extranuclear or nongenomic effects of thyroid hormone are unaffected by the inhibitors of protein synthesis, and their time course cannot be explained by the interaction of the hormone molecule with nuclear receptors. Their site of action has been localized at the plasma membrane, but also at the cytoplasm and organelles such as the mitochondria. In particular thyroid hormone has been reported to activate, by both genomic and non genomic mechanism, the Ca2+-ATPase, an ion pump that removes calcium from the cytosol and stores it in the sarcoplasmic reticulum. The decrease in intracellular Ca2+ generated during the systole leads to cardiac muscle relaxation. Given to all these important effects on the cardiovascular system, T3 can be also envisaged as a potent novel therapeutic agent as a inotropic drug. Thyroid hormone is also a major regulator of the Na/K-ATPase activity in several tissues. L-T3 and its analog 3,5-diiodothyronine give rise to a fast inhibition of the Na+/K+-ATPase activity in chick embryo hepatocytes, whereas L-T4 appears to be ineffective. The Na/H exchanger, activated by the thyroid hormone by both genomic and nongenomic mechanism, is a ubiquitous plasma membrane integral protein exchanging extracellular Na+ with cytoplasmic H+ ions according to the concentration gradient; it does not require energy supply, but depends on the Na+/K+-ATPase. Some of these effects are mediated by PKC and MAPK pathway, likely interfaces between genomic and nongenomic effects of thyroid hormones. The extranuclear effects should be taken into account when considering appropriate therapeutic intervention based on thyroid hormone both for their possible reinforcement of the nuclear effects and for their fast time onset, that might be of clinical relevance particularly for the cardiovascular system

Thyroid Hormones. From Molecular Signaling to Human Disease: An Update

This Thematic Issue presents reviews of recent studies on effects of thyroid hormones and certain metabolites that are linked to human diseases, providing a general update of the topic. I have been the guest editor for thematic issues of IEMAMC for three times, in 2006, 2011 and 2015, and over these 10 years my knowledge of the nongenomic effects of thyroid hormone has improved considerably. First issue was dedicated to the nongenomic effects of various hormones endowed with classical nuclear receptors, and included a chronology of the earliest reported nongenomic effects of thyroid hormones. Second issue was centered on thyroid hormone nongenomic mechanisms in skeletal muscle and nerve cells, mainly, but not exclusively, dealing with effects mediated by integrin alphaVbeta3. In the current issue, the coverage of thyroid hormone actions is wider, including also nuclear classical effects, the cognitive processes and effects of some hormone metabolites that are particularly interesting from pharmacological point of view. Why? When a new surprising discovery is published, such as the seminal paper by Bergh et al., in 2005, showing that the integrin alphaVbeta3 was the long-searched plasma membrane receptor for thyroid hormones, it can of course dominate the scenery for years, and other researchers in this field will use this new piece of information in their studies to understand the physio-pathological effects of thyroid hormones. The Bergh et al. paper showed that binding by thyroxine to the integrin alphaVbeta3 gives rise to a downstream signaling that causes MAPK activation and tumor cell proliferation. This has so to say been the ‘Big Bang’ of nongenomic effects of thyroid hormones. After ten years of the integrin story, we can now observe how many things have changed in our knowledge about the role of thyroid hormones in animal physiology. However, actually the integrin story began before alphaVbeta3 was recognized as a plasma membrane thyroid hormone receptor. The group of Davis and Davis showed, in 2004, that various effects of thyroid hormone on the actions of epidermal growth factor and transforming growth factor-alpha were mediated by cAMP-dependent protein kinase II. The crosstalk between thyroid hormones and growth factors reported in that paper would eventually lead to the integrin; alphaVbeta3 was ‘in nuce’ although it was not clear at that time. Now, this interaction of thyroid hormones with growth factors is coming of age and may lead us far, to different and still unknown aspects of physiology and pathology, as it can be observed from some of the contributions in this issue

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.

"tAtrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in theimmune system. The aim of the present study was to investigate in THP-1 monocytes the ANP effecton 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 ofANP (10−10M). The ANP action was partially affected by cell pretreatment with PD98059, an inhibitor ofmitogen activated-protein kinases (MAPK) as well as by wortmannin, an inhibitor of phosphatidylinositol3-kinase (PI3K) and totally suppressed by diphenylene iodonium (DPI), an inhibitor of the enzyme nico-tinamide adenine dinucleotide phosphate (NADPH) oxidase. The hormone effect was mimicked by cANFand an ANP\/NPR-C signaling pathway was studied using pertussis toxin (PTX). A significant increase ofH2O2-induced cell migration was observed after ANP (10−10M) treatment, conversely a decrease of THP-1proliferation, due to cell death, was found. Both ANP actions were partially prevented by DPI. Moreover,H2O2-induced release of IL-9, TNF-, MIP-1 and MIP-1 was not counteracted by DPI, whereas no effectwas observed in any experimental condition for both IL-6 and IL-1. Our results support the view thatANP can play a key role during the inflammatory process.