Search

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.

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. The effect of l-T4 was blocked in the presence of the 5′-deiodinase inhibitor 6-n-propyl-2-thiouracil, suggesting that the difference in lag time between l-T3 and l-T4 was due to the 5′-deiodination process that transforms l-T4 into the bioactive l-T3. In short term studies (

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. The transphosphatidylation reaction, as well as its labeling ratio, demonstrate that phosphatidylcholine-dependent phospholipase D (PC-PLD) is not involved. Our experiments with R59022, a DAG kinase (DAGK) inhibitor, indicate that such an increase may be due to the phosphorylation of DAG derived from phosphatidylinositol (PI) hydrolysis. Our results show that phorbol 12-myristate 13 acetate (PMA) plays a significant role in late DAG formation and that Pcho is released concomitantly, suggesting there is a relationship between the two phospholipase Cs (PLCs) that occurs through a protein kinase C (PKC) translocation from cytosol to the plasma membrane. These findings are confirmed by the use of PKC inhibitors calphostin, H7, and staurosporine. The involvement of membrane phospholipid hydrolysis and the ensuing production of second messengers might explain the vasorelaxant effect of ANF.

Extranuclear or nongenomic actions of thyroid hormones are unaffected by the inhibitors of protein synthesis, their site of action has been localized at the plasma membrane but also in the cytoplasm and organelles such as the mitochondria. This review takes into account recent major advances in nongenomic effects of thyroid hormones in nervous system, immune system and cardiovascular tissue, with a particular focus on the plasma membrane receptor integrin αvβ3. In nerve cells nongenomic effects of thyroid hormones point mainly to a direct modulation of several channels/receptors for the major neurotransmitters, even though more complex pathways have also been demonstrated. Certain neuroprotective actions have recently been described for thyronamines, and this may be relevant to Alzheimer's disease and multiple sclerosis. The immune system is also modulated nongenomically by thyroid hormones, through potentiation of the effects of cytokines such as IFN-γ or lipopolysaccharide, or through activators of STAT protein leading to activation of the mammalian target of rapamycin (mTOR) pathway, a highly conserved kinase downstream target of nongenomic actions of thyroid hormone. The mTOR system is also involved in the cardioprotection mechanisms, where thyroid hormone signaling through the receptor integrin αvβ3 may play an important role that needs to be further studied. The identification of integrin αvβ3 as a plasma membrane receptor for thyroid hormones has provided a new perspective on the role of these hormones in cellular defense. Analogs of thyroid hormones, inhibitors and agonists at the integrin receptor for the hormone and mTOR inhibitors are evaluated as areas of emphasis for therapeutic research.

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 (H 2 O 2 )-induced Reactive Oxygen Species (ROS), cell proliferation and migration. A significant increase of H 2 O 2 -dependent ROS production was induced by physiological concentration of ANP (10 −10 M). The ANP action was partially affected by cell pretreatment with PD98059, an inhibitor of mitogen activated-protein kinases (MAPK) as well as by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) and totally suppressed by diphenylene iodonium (DPI), an inhibitor of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The hormone effect was mimicked by cANF and an ANP/NPR-C signaling pathway was studied using pertussis toxin (PTX). A significant increase of H 2 O 2 -induced cell migration was observed after ANP (10 −10 M) treatment, conversely a decrease of THP-1 proliferation, due to cell death, was found. Both ANP actions were partially prevented by DPI. Moreover, H 2 O 2 -induced release of IL-9, TNF-α, MIP-1α and MIP-1β was not counteracted by DPI, whereas no effect was observed in any experimental condition for both IL-6 and IL-1β. Our results support the view that ANP can play a key role during the inflammatory process.

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 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.

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. Sticholysin II (StnII) is a 20 kDa protein from the sea-anemone Stichodactyla helianthus which shows a cytotoxic activity by forming oligomeric aqueous pores in the cell plasma membrane. A recent NMR analysis indicates that the sticholysin II binds specifically to sphingomyelin by two domains that recognize respectively the hydrophilic (i.e. phosphorylcholine) and the hydrophobic (i.e. ceramide) moieties of the molecule. Aim of our research has been to verify the possible employ of an antibody against the StnII to investigate the localization and the dynamics of sphingomyelin in cell membranes. For this purpose, we developed a monoclonal antibody (named A10) against the toxin and we tested its ability to bind StnII after binding to sphingomyelin. A10 antibody is able to recognize the sticholysin II both in its native form and after SDS treatment, being the protein still suitable for many analytic techniques such as ELISA, western blotting and immunofluorescence. The high affinity of the toxin for the sphingomyelin in cell membranes has been demonstrated by microscopic immuno-localization and western blot analysis; both methods confirmed that sphingomyelin is the molecular acceptor for StnII also in cell membranes. Finally, we studied the specificity of the toxin for sphingomyelin by a cell membrane-double labelling method, using cholera toxin, specific for the ganglioside GM1, and sticholysin II. The results obtained show that there is no cross-reactivity between the two toxins, confirming that sticholysin II is able to discriminate among membrane domains with sphingomyelin with respect to those enriched with gangliosides.

""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.. ""

The signals initiated by skeletal muscle contraction and exercise involve several biochemical mechanisms both in the short- and long-time range that are under the control of thyroid hormones as genomic as well as nongenomic mechanisms. The effect of thyroid hormones on skeletal muscle has been studied both in cells in culture and in vivo. In L- 6 myoblasts thyroid hormone increases intracellular calcium and pH. Administration of a single injection of thyroid hor- mone in rat i.p. leads to a rapid rise in T3 about 50-fold, that peaked at 2 hours. The effect of this treatment was evaluated on signaling kinases, in particular on the phosphorylation of the p38MAPK pathway, and AMP-activated protein kinase. Both were activated and both can be a downstream target of the integrinv� 3, the reported plasma membrane receptor for thyroid hormones. Contractile activity leads to mitochondrial biogenesis within skeletal muscle, which gives rise to sig- naling pathways leading to the expression of transcription factors. These are activated by a Ca 2+ -dependent release, p38 and AMP-activated protein kinase. The activated transcription factors such as nuclear respiratory factor 1, interact with the coactivator PGC-1� to stimulate the expression of nuclear genes encoding mitochondrial proteins. This sequence of events can be induced not only by physical exercise, but also by thyroid hormone (T3) treatment in vivo, particularly in slow twitch skeletal muscle. In conclusion, the old statement that thyroid hormone helps to keep the steady state, is still valid, furthermore it contributes to a good performance of skeletal muscle as exercise does. + /H + ex- changer, p38MAPK, peroxisome proliferator-activated receptorcoactivator-1� , physical exercise, skeletal muscle, thyroid hormones.

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.

It is widely accepted that insulin action does not involve inositol phospholipid hydrolysis through the stimulation of a phosphatidylinositol-specific phospholipase C (PI-PLC). This consideration prompted us to investigate the insulin effect on the mechanism leading to the accumulation of diacylglycerol (DAG) and phosphatidic acid (PA) in rat hepatocytes. Basically, insulin induces; (i) a significant increase of both [3H]glycerol and fatty acid labelling of DAG; (ii)_a significant increase of PA labelling preceeding DAG labelling and paralleled by a decrease of phosphatidylcholine (PC) labelling. These observations, which suggest an insulin-dependent involvement of a phospholipase D, are strengthened by the increase of PC-derived phosphatidylethanol in presence of ethanol. Finally, the observation that the PA levels do not return to basal suggests that other mechanisms different from PC hydrolysis, such as the stimulation of direct synthesis of PA, may be activated.

The relationship between cell proliferation and inositol lipid turnover has been studied by comparing the steady state of inositol derivative metabolism in quiescent and regenerating rat hepatocytes isolated at 4 h (G1 phase of first cell cycle) and 24 h (onset of M phase) after partial hepatectomy. The effect of two hormones able to regulate hepatic regeneration, insulin and vasopressin, has been considered, and the results can be summarized as follows: (i) at 4 h after partial hepatectomy, the precursor incorporation into inositol polyphosphates and the particulate phospholipase C activity increase with respect to quiescent hepatocytes, whereas the content of I1,4,5P3 does not change, suggesting an increased turnover of this molecule in this step of cell cycle priming; (ii) 24 h after partial hepatectomy, the radioactivity linked to IP3 and IP4, as well as soluble and particulate phospholipase C activity, and IP3 content increase, suggesting the presence, at the onset of M phase, of second messenger accumulation; (iii) only 24 h after partial hepatectomy, the inositol derivative metabolism is affected by vasopressin; and (iv) insulin exerts a modulatory role on inositol polyphosphate production without involving membrane-bound PLC activity or phosphoinositide hydrolysis. These data suggest that inositol-derived signal molecules are associated with hepatic regneration; moreover, the metabolic pathway of such compounds seems to be regulated so that only specific inositol phosphates are present in each step of the cell cycle. © 1992 Wiley-Liss, Inc.

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.

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. These results suggest that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, at subtoxic concentrations, might synergize with scarcely toxic doxorubicin doses to propagate a caspase-independent cytotoxic response, such that P-glycoprotein-dependent drug resistance is circumvented.

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). Results obtained demonstrate that MbetaCD prevents the changes induced by anoxic insult, i.e., depression of the population spike amplitude and insurgence of ischemic long-term potentiation. Furthermore cholesterol depletion prevents the disappearance of population spike induced by anoxia/aglycemia during kainate perfusion. Our data suggest a possible role of MbetaCD in preventing the pathological changes in synaptic activity induced by ischemia and indicate that manipulation of lipid components of membrane rafts might provide a new approach for the treatment of ischemia.

The capacity of the snake venom neurotoxin beta-bungarotoxin to induce fusion of small unilamellar liposomes was demonstrated. The fusion process was studied spectroscopically using three different methods: (i) by resonance energy transfer, using fluorescent lipid analogues; (ii) by the Tb/DPA assay; (iii) by electron spin resonance, using spin-labeled phospholipids. For the latter technique, a new method of analysis based on Fourier-transform component separation was developed. The fusogenic activity was found to be strongly correlated with the known phospholipase A2 activity of the toxin: both functions were shown to have a specific requirement of Ca2+ at almost stoichiometrical concentrations, much below the threshold values found for unspecific divalent cation induced vesicle fusion. Similarly, the presence of phosphatidic acid in the target membrane was essential for both fusogenic and enzymatic activities. The results suggest a molecular mechanism of fusion involving protein binding to negatively charged groups on the membrane surface, followed by local formation of lysophospholipids and as a consequence hereof the creation of point defects in the lipid structure. On the basis of these findings, a model is put forward to explain the specific mode of action of beta-bugarotoxin in vivo.

Liver lipid composition of the frogRana esculenta was examined on a circannual basis. In particular, phospholipid and cholesterol content, relative phospholipid distribution, and fatty acid patterns have been studied. Seasonal acclimatization is associated with significant modifications of phospholipid content and of the relative proportion of phospholipid classes, while cholesterol level is unchanged throughout the year. In regard to the fatty acid composition of total phospholipids as well as of the four major phospholipid classes—phosphatidylcholine (PC); phosphatidylethanolamine (PE); sphingomyelin (SM); phosphatidylserine (PS)—it appears that the liver of “summer animals” is characterized by a higher unsaturation index due to a decrease of saturated fatty acids and to an increased content of n−3 and n−6 polyunsaturated fatty acids. The results suggest that relevant compositional changes occur mainly in spring and autumn: these changes could be interpreted as being the result of both a nutritionally- and thermally-induced seasonal adaptation directed toward the preservation of membrane-associated physiological activities that are linked to the transition from the active to the inactive state of the animal.

Atrial natriuretic peptide induces cell death in human hepatoblastoma (HepG2) through the involvement of NADPH oxidase

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. A significant decrease of pHi was observed in ANP-treated macrophages with respect to untreated cells; this effect was paralleled by enhanced phospholipase activity and ROS production. Moreover, all assessed ANP effects seem to be mediated by the NPR-C. In contrast, no significant effect on pHi was observed in THP-1 monocytes treated with ANP. Treatment of macrophages or THP-1 monocytes with 5-(N-ethyl-N-isopropyl)amiloride, a specific Na+/H+ antiport inhibitor, decreases pHi in macrophages and monocytes. Our results indicate that only macrophages respond to ANP in terms of pHi and ROS production, through diacylglycerol and phosphatidic acid involvement, pointing to ANP as a new modulator of ROS production in macrophages.

Atrial natriuretic factor (ANF) is a polypeptide able to affect cardiovascular home-ostasis 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. The potential antimitogenic role of ANF on the cell growth induced by growth factors appears very intriguing. Aim of the present study was to investigate the possible involvement of ANF on rat aortic smooth muscle (RASM) cells proliferation induced by known mitogens and the mechanism involved. Our data show that ANF, at physiological concentration range, inhibits RASM cell proliferation induced by known mitogens such as PDGF and insulin, and the effect seems to be elicited through the modulation of phosphatidic acid (PA) production and MAP kinases involvement. (C) 2002 Wiley-Liss, Inc.

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.

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. Recent experiments on living cells do not confirm this hypothesis, and a more intricate mechanism is proposed. In vitro, ammodytin L has necrotic effects only in well-differentiated myogenic cells, whereas other cell types such as platelets, red blood cells and lymphocytes show neither morphological nor functional alterations. In this work we demonstrate that rat 208F fibroblasts in culture after ammodytin L challenge increase [3H]thymidine incorporation, indicating that this toxin has a myogenic effect. Moreover, ammodytin L increases intracellular Ca2+ by acting on intracellular stores probably by activating a phosphatidylinositol-specific phospholipase C. Preincubation of the cells with ammodytin L did not prevent the massive Ca2+ release evoked by bradykinin, a phenomenon observed when fibroblasts were incubated with both thapsigargin and ionomycin. Heparin, an agent that inhibits the necrotic effect of the myotoxin in myotubes, also reduces the effect of ammodytin L on DNA synthesis. Heparin inhibits only the late sustained increase in intracellular Ca2+ induced by the toxin.

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. Only muscular cells were affected by ammodytin L, and the toxin did not induce any morphological change in other cell types. Further evidence of the muscle-specific action of the toxin was obtained from experiments carried out using the Xenopus kidney cell line B3.2 in culture. Ammodytin L was unable to affect parameters of cell viability such as lactate dehydrogenase leakage, [3H]thymidine incorporation, growth curves and morphological changes. Moreover, direct ammodytin L application to cultured regenerative limbs did not provoke alterations in undifferentiated myoblasts. These data suggest that ammodytin L, like other phospholipase-like toxins, exerts its toxicity by selectively damaging differentiated muscle fibres.

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.

Muscarinic stimulation of the human neuroblastoma cell line SK-N-BE(2) elicits hydrolysis of phosphoinositides and phosphatidylcholine (PtdCho) and produces a rapid and sustained elevation of diacylglycerol (DG) mass. PtdIns(4,5)P2 cleavage by phospholipase C (PLC) occurred immediately after carbachol (CCh) addition, and phosphoinositide hydrolysis was then sustained for at least 5 min. Cell stimulation, after extensive PtdCho labelling by long-term [3H]choline administration, resulted in an enhanced release of [3H]phosphocholine (PCho) into the external medium; enhanced [3H]PCho release, which occurred with a 15 s delay with respect to CCh addition, was particularly pronounced within the first minute of stimulation and proved to be caused by PtdCho-specific PLC activation. In fact, when cells were exposed to [3H]choline for a short period, to extensively label the intracellular PCho pool but not PtdCho, stimulation did not result in an enhanced release of [3H]PCho into the medium. PtdCho-specific phospholipase D (PLD) activation was documented by the accumulation of [3H]phosphatidylethanol in cells prelabelled with [3H]myristic acid and stimulated in the presence of 1% (v/v) ethanol; this metabolic pathway, however, proved to be a minor one leading to generation of phosphatidic acid (PtdOH) during cell stimulation, whereas DG production by the sequential action of PtdCho-specific PLD and PtdOH phosphohydrolase was not observed. Studies on cells which were double-labelled with [3H]myristic acid and [14C]arachidonic acid indicated that within 15 s of stimulation DG is uniquely derived from PtdIns(4,5)P2, whereas PtdCho is the major source at later times. Evidence is provided that rapid and selective conversion of phosphoinositide-derived DG into PtdOH may play an important role in determining the temporal accumulation profile of DG from the above-mentioned sources.

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.

Mouse keratinocytes cultured in a medium containing less than 0.1 mM Ca 2+ (low Ca 2+ ) incorporated [1− l4 C]arachidonic acid (AA) into phospholipids by kinetics including; (i) a rapid labelling of phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and both acid-stable and alkenylacyl forms of phosphatidylcholine (PtdCho); and (ii) a slow but long-lasting radiolabel incorporation into both acid-stable and alkenylacyl forms of phosphatidylethanolamine (PtdEtn), partly associated with a net radioactivity loss from acid stable-PtdCho. Under low Ca 2+ conditions no radioactivity transfer apparently occurred between PtdIns and other phospholipid classes. When cells were prelabelled for 24 h with [1− 14 C]AA and reincubated in label-free medium containing 1.2 mM Ca 2+ (normal Ca 2+ ), an early and extensive loss of radioactivity from PtdIns was observed, reasonably in connection with Ca 2+ stimulation of phosphoinositide turnover [1]. Cell shift to normal Ca 2+ did not result in an increased synthesis of labelled eicosanoids, but was consistent with an increase of radioactivity incorporation into diacylglycerol (DAG) and with a complex pattern of [1− 14 C]AA redistribution, eventually leading to a marked radioactivity incorporation into acid stable-PtdEtn (but not into alkenylacyl-PtdEtn) and to a labelling decrease of acid stable-PtdCho. The possible mechanisms driving AA recycling after cell shift to normal Ca 2+ are discussed.

The incorporation and redistribution of [1-14C]arachidonic acid in SK-N-BE human neuroblastoma cell phospholipids were investigated. By continuous labelling in serum-enriched medium, a rapid radioactivity incorporation into phosphatidylcholine (PtdCho), phosphatidylinositol, and phosphatidylserine was observed; initially, phosphatidylethanolamine (PtdEtn) was poorly labelled, but at later stages it displayed the highest level of arachidonic acid incorporation, in comparison with other phospholipid classes. Labelling of triacylglycerols was also observed. When cells were pulse-labelled with [1-14C]arachidonic acid and then reincubated in label-free medium, a decrease of the radioactivity in triacylglycerols was observed initially, paralleled by an increase of phospholipid labelling; thereafter, arachidonic acid redistribution was consistent with a net decrease of the radioactivity associated with PtdCho acid-stable forms (i.e., diacyl plus alkylacyl forms), concomitantly with a net labelling increase of both acid-stable PtdEtn and alkenylacyl-PtdEtn. Data indicate the following: (a) neuroblastoma cells incorporate arachidonic acid into phospholipids through complex kinetics involving transfer of the fatty acid from acid-stable PtdCho to both alkenylacyl-PtdEtn and acid-stable PtdEtn; and (b) triacylglycerols act as storage molecules for arachidonic acid which is subsequently incorporated into phospholipids. The possibility that arachidonic acid transfer to PtdEtn subclasses is driven by distinct mechanisms is discussed.

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.

The hormonal responsiveness of plasma membrane-bound enzymes (Na-+-K-+)-ATPase and adenylate cyclase has been investigated in normal and regenerating rat liver. (Na-+-K-+)-ATPase basal activity is not affected by surgery and only slightly affected by partial hepatectomy; its response to epinephrine and cyclic AMP is decreased only 15 h after hepatectomy. Adenylate cyclase activity of plasma membranes from untreated animals is stimulated by parathyroid hormone and thyroxine; partial hepatectomy increased basal activity as well as the stimulation exerted by the aforementioned hormones, when glucagon and epinephrine sensitivity is essentially unaltered.

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.

The effect of twelve l -amino acids on the activity of liver plasma membrane (Na+K+)-ATPase has been tested. Histidine and arginine significantly enhanced the activity. The activtion by histidine showed saturation kinetics with an apparent Ka of about 8 mM, and was evident over a wide range of Na+ concentrations. The same amino acid did not significantly affect the Mg2+-dependent ATPase activity.

Adenosine 3′-,5′-cyclic monophosphate phosphodiesterase (EC 3.1.4.17) has been investigated in rat liver as to its insulin sensitivity. Hormone action has been assayed in vitro on a liver hemogenate purified by DEAE-cellulose column chromatography, on isolated hepatocytes, on isolaetd plasma membranes. The DEAE-cellulose chromatography purified homogenate showed no sensitivity to insulin, whereas isolated hepatocytes incubated in presence of insulin showed increased phosphodiesterase activity in a plasma membrane-containing fraction. The plasma membrane-bound enzyme, which shows both high and low affinity components, was significantly stimulated after hormonal treatment; this effect being dependent on a V increase of the low K m form.

The effect of cyclic AMP on (Na+—K+)-ATPase and protein kinase activities of isolated liver plasma membrane has been investigated. Theophylline and caffeine, several cyclic nucleotides (including cyclic AMP, dibutyryl cyclic AMP, cyclic GMP and cyclic UMP), and sodium butyrate reduced the activity of (Na+—K+)-ATPase. The most powerful inhibitor was dibutyryl cyclic AMP; however, its action is probably due to the combined effects of cyclic AMP and butyrate. The blocking agent propranolol prevented the action of both epinephrine and cyclic AMP. Liver plasma membranes contain protein kinase(s), which catalyze the transfer of terminal phosphate of ATP into protein of plasma membrane itself (endogenous phosphorylation) as well as into added proteins (exogenous phosphorylation). Endogenous phosphorylation was inhibited by cyclic AMP, dibutyryl cyclic AMP and cyclic GMP. On the other hand, exogenous phosphorylation was stimulated by cyclic AMP. The specific activity of the membrane enzyme catalyzing the latter reaction was found to be higher than that of the enzyme present in the 100 000 × g supernatant of rat liver. The parallel effect of cyclic AMP on (Na+—K+)-ATPase and on the phosphorylation of membrane protein, and the finding that the latter reaction involves the formation of acylphosphate bonds, may be indicative of a possible role of endogenous phosphorylation in the regulation of (Na+—K+)-ATPase activity.

The chemical composition of liver plasma membrane was studied in Wistar rats aged between 3 and 24 months. Results obtained indicate a significant age-dependent positive correlation of both the protein: phospholipid and cholesterol: phospholipid ratios, whereas the protein: cholesterol ratio seems to remain unaffected. Phospholipid analysis of liver plasma membrane reveals that only the phosphatidylcholine content has a significant negative correlation with age; all other phospholipid species remain basically unchanged.

The effect of cholesterol and fatty acid treatment in vitro was tested on rat liver plasma membrane-bound enzymes and lipid fluidity. The observed alterations of membrane fluidity affect both (Na+−K+)-ATPase and Mg2+-ATPase activities but not 5′-nucleotidase; basal adenylate cyclase as well as its hormonal sensitivity were differentially affected by changes of membrane microenvironment.

The effect of insulin on human red blood cells was investigated, both on intact cells and on isolated plasma membranes, testing the responsiveness of membrane-bound enzymes--such as (Na+-K+)-ATPase and 5'-nucleotidase--as well as the ouabain binding and ionic fluxes. It appears that insulin stimulates Na-pumping mechanisms increasing (Na+-K+)-ATPase activity through an enhanced availability of pumping sites, as can be inferred from the increased ouabain binding. The apparent unresponsiveness of fluorescence polarization parameters, following insulin treatment of isolated plasma membranes and intact cells, rules out--at present--an involvement of membrane lipid fluidity in the mechanism of action of insulin on human erythrocytes.

The Na+-pumping activity of the erythrocyte plasma membrane in diabetic subjects was studied together with the lipid composition. Insulin-dependent diabetes mellitus (IDDM) patients (n = 25) were divided into young (28.1 ± 7.4 yr old, mean ± SD; n = 16) and old (71.7 ± 9.8 yr old; n = 10) subjects; the age of non-insulin-dependent (NIDDM) patients was 70.7 ± 11.5 yr (n = 10). The Na+-pumping activity, estimated from both Na+-K+-ATPase and ouabain binding, was significantly decreased in IDDM and NIDDM subjects, but its insulin sensitivity was retained only in young IDDM subjects. The total cholesterol and phospholipid content of the erythrocyte plasma membrane was lowered in IDDM subjects, and cholesterol-to-phospholipid molar ratio was significantly decreased. In NIDDM subjects the significant decrease of the two lipid components did not alter their ratio. The analysis of major phospholipid components of erythrocyte membranes revealed that only phosphatidylcholine is significantly increased in young diabetic subjects. The fatty acid composition of major phospholipid classes was significantly altered in all cases: the unsaturation index appeared to be increased in phosphatidylserine and sphingomyelin for both IDDM and NIDDM subjects and was also increased in phosphatidylcholine in the latter group.