Tomás Gómez

Biophysical Alterations in Lipid Rafts from Human Cerebral Cortex Associate with Increased BACE1/AβPP Interaction in Early Stages of Alzheimer's Disease

In the present study, we have assessed the biophysical properties of lipid rafts from different brain areas in subjects exhibiting early neuropathological stages of Alzheimers disease (AD). By means of steady-state fluorescence polarization analyses using two environment-sensitive fluorescent probes, we demonstrate that lipid rafts from cerebellum, and frontal and entorhinal cortices, exhibit different biophysical behaviors depending on the stage of the disease. Thus, while membrane anisotropies were similar in the cerebellum along stages, lipid rafts from frontal and entorhinal cortices at AD stages I/II and AD III were significantly more liquid-ordered than in control subjects, both at the aqueous interface and hydrophobic core of the raft membrane. Thermotropic analyses demonstrated the presence of Arrhenius breakpoints between 28.3-32.0 °C, which were not influenced by the disease stage. However, analyses of membrane microviscosity (ηapp) demonstrate that frontal and entorhinal lipid rafts are notably more viscous and liquid-ordered all across the membrane from early stages of the disease. These physicochemical alterations in lipid rafts do not correlate with changes in cholesterol or sphingomyelin levels, but to reduced unsaturation index and increased saturate/polyunsaturated ratios in phospholipid acyl chains. Moreover, we demonstrate that β-secretase/AβPP (amyloid-β protein precursor) interaction and lipid raft microviscosity are strongly, and positively, correlated in AD frontal and entorhinal cortices. These observations strengthens the hypothesis that physical properties of these microdomains modulate the convergence of amyloidogenic machinery toward lipid rafts, and also points to a critical role of polyunsaturated fatty acids in amyloidogenic processing of AβPP.

Acute relaxation of mouse duodenun by estrogens: Evidence for an estrogen receptor-independent modulation of muscle excitability

17-beta-Estradiol, the stereoisomer 17-alpha-estradiol and the synthetic estrogen diethylstilbestrol (DES), all caused a rapid (<3 min) dose-dependent reversible relaxation of mouse duodenal spontaneous activity, reduced basal tone and depressed the responses to CaCl(2) and KCl. The steroidal antiestrogen 7alpha-[9-[(4,4,5,5,5,-pentafluoropenty)sulphinyl]nonyl]-estra-1,3,5(19)-triene-3,17beta-diol (ICI182,780) failed to either mimic or prevent the effect of 17-beta-estradiol. The effect of estrogens was unrelated to activation of nitric oxide (NO), mitogen-activated protein kinase (MAPK), protein kinase A (PKA), protein kinase G (PKG) or protein kinase C (PKC). Estrogen-induced relaxation was partially reversed by 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-pyridine-3-carboxilic acid methyl ester (BAY-K8644), depolarization, or by application of tetraethylammonium or 4-aminopyridine, but not by glibenclamide, apamin, charybdotoxin, paxilline or verruculogen. The effects of BAY-K8644 and K(+) channel blockers were synergistic, and allowed relaxed tissues to recover spontaneous activity and basal tone. We hypothesize that the rapid non-genomic spasmolytic effect of estrogens on mouse duodenal muscle might be triggered by an estrogen-receptor-independent mechanism likely involving activation of tetraethylamonium- and 4-aminopyridine-sensitive K(+) channels and inhibition of L-type Ca2(+) channels on the smooth muscle cells.

Androgens are powerful non-genomic inducers of calcium sensitization in visceral smooth muscle

Androgens are recognized as genotropic inducers of a number of physiological functions mainly associated with the development of sexual characteristics. However, as in the case of estrogens, the number of studies evidencing androgen actions in non-reproductive tissues has steadily grown over the past years. Here, we show that androgens acutely ( approximately 30min) alter the frequency spectrum of peristaltic activity of intestinal smooth muscle and augment the amplitude agonist-induced contractile activity. Maximal stimulation occurred at physiological concentrations of androgens with EC(50) values in the picomolar range. Androgen-induced potentiation was prevented by preincubation with androgen receptor (AR) antagonists but unaffected by cycloheximide plus actinomycin D, indicating that potentiation was mediated by ARs via a non-genomic mechanism. The effects of androgens were mimicked by polyamines and were completely blocked by inhibitors of polyamine synthesis. Using ionomycin-permeabilized intestinal smooth muscle preparations, we demonstrate that androgens exert their effects by inducing a mechanism of sensitization to calcium and not by altering intracellular calcium homeostasis. Correspondingly, the potentiation of mechanical activity induced by androgens was accompanied by an increase in the phosphorylation of the regulatory myosin light chain (LC(20)) within the same time-course than calcium sensitization and mechanical potentiation. The pursuit of potential signalling pathways linking androgen receptor activation with calcium sensitization revealed that mechanical potentiation of intestinal muscle by androgens involve activation of the Rho pathway, whose downstream effector, Rho-associated kinase (ROCK), is eventually responsible for displacement of the phosphorylation/dephosphorylation state of LC(20) towards its phosphorylated form.

Androgens Induce Nongenomic Stimulation of Colonic Contractile Activity through Induction of Calcium Sensitization and Phosphorylation of LC20 and CPI-17

We show that androgens, testosterone and 5alpha-dihydrotestosterone (DHT), acutely (approximately 40 min) provoke the mechanical potentiation of spontaneous and agonist-induced contractile activity in mouse colonic longitudinal smooth muscle. The results using flutamide, finasteride, cycloheximide, and actinomycin D indicate that androgen-induced potentiation is dependent on androgen receptors, requires reduction of testosterone to DHT, and occurs independently of transcriptional and translational events. Using permeabilized colonic smooth muscle preparations, we could demonstrate that mechanical potentiation is entirely due to calcium sensitization of contractile machinery. In addition, DHT (10 nm) increased phosphorylation of both 20-kDa myosin light chain (LC(20)) [regulatory myosin light chain, (MLC)] and CPI-17 (an endogenous inhibitor of MLC phosphatase). Paralleling these findings, inhibition of Rho-associated Rho kinase (ROK) and/or protein kinase C (PKC) with, respectively, Y27632 and chelerythrine, prevented LC(20) phosphorylation and abolished calcium sensitization. In addition, inhibition of ROK prevents CPI-17 phosphorylation, indicating that ROK is located upstream PKC-mediated CPI-17 modulation in the signalling cascade. Additionally, androgens induce a rapid activation of RhoA and its translocation to the plasma membrane to activate ROK. The results demonstrate that androgens induce sensitization of colonic smooth muscle to calcium through activation of ROK, which in turn, activates PKC to induce CPI-17 phosphorylation. Activation of this pathway induces a potent steady stimulation of LC(20) by inhibiting MLC phosphatase and displacing the equilibrium of the regulatory subunit towards its phosphorylated state. This is the first demonstration that colonic smooth muscle is a physiological target for androgen hormones, and that androgens modulate force generation of smooth muscle contractile machinery through nongenomic calcium sensitization pathways.

Mechanisms of transport of Na+ and Cl− in the lizard colon

1. Ionic fluxes of sodium and chloride across lizard colon mucosa were measured and compared with the electrical characteristics of the tissue under voltage-clamped conditions. 2. In a Ringer-bicarbonate solution there was both a net sodium flux (JNanet) and a net chloride flux (JClnet) from mucosa to serosa. The net flux residual (JR) was near zero, indicating that net sodium and chloride transport is the result of an electrically neutral transport mechanism. 3. In the presence of sodium, the net chloride flux was abolished and the short-circuit current (Isc) and the electrical potential difference (PD) were unchanged. In the absence of chloride the net sodium flux was abolished and the short-circuit current and electrical potential difference were not modified. 4. From an analysis of the effects of the inhibitors, furosemide, amiloride and disulfonic stilbene (DIDS), a plausible model was developed to explain the characteristics of sodium and chloride absorption.

Unique SERM-like properties of the novel fluorescent tamoxifen derivative FLTX1.

Tamoxifen is a selective estrogen receptor modulator extensively used on estrogen receptor-positive breast cancer treatment. However, clinical evidences demonstrate the increased incidence of undesirable side effects during chronic therapies, the most life threatening being uterine cancers. Some of these effects are related to tissue-dependent estrogenic actions of tamoxifen, but the exact mechanisms remain poorly understood. We have designed and synthesized a novel fluorescent tamoxifen derivative, FLTX1, and characterized its biological and pharmacological activities. Using confocal microscopy, we demonstrate that FLTX1 colocalizes with estrogen receptor α (ERα). Competition studies showed that FLTX1 binding was totally displaced by unlabeled tamoxifen and partially by estradiol, indicating the existence of non-ER-related triphenylethylene-binding sites. Ligand binding assays showed that FLTX1 exhibits similar affinity for ER than tamoxifen. FLTX1 exhibited antiestrogenic activity comparable to tamoxifen in MCF7 and T47D cells transfected with 3xERE-luciferase reporter. Interestingly, FLTX1 lacked the strong agonistic effect of tamoxifen on ERα-dependent transcriptional activity. Additionally, in vivo assays in mice revealed that unlike tamoxifen, FLTX1 was devoid of estrogenic uterotrophic effects, lacked of hyperplasic and hypertrophic effects, and failed to alter basal proliferating cell nuclear antigen immunoreactivity. In the rat uterine model of estrogenicity/antiestrogenicity, FLTX1 displayed antagonistic activity comparable to tamoxifen at lower doses, and only estrogenic uterotrophy at the highest dose. We conclude that the fluorescent derivative FLTX1 is not only a suitable probe for studies on the molecular pharmacology of tamoxifen, but also a potential therapeutic substitute to tamoxifen, endowed with potent antiestrogenic properties but devoid of uterine estrogenicity.

Hippocampal Lipid Homeostasis in APP/PS1 Mice is Modulated by a Complex Interplay Between Dietary DHA and Estrogens: Relevance for Alzheimer's Disease

Current evidence suggests that lipid homeostasis in the hippocampus is affected by different genetic, dietary, and hormonal factors, and that its deregulation may be associated with the onset and progression of Alzheimers disease (AD). However, the precise levels of influence of each of these factors and their potential interactions remain largely unknown, particularly during neurodegenerative processes. In the present study, we have performed multifactorial analyses of the combined effects of diets containing different doses of docosahexaenoic acid (DHA), estrogen status (ovariectomized animals receiving vehicle or 17β-estradiol), and genotype (wild-type or transgenic APP/PS1 mice) in hippocampal lipid profiles. We have observed that the three factors affect lipid classes and fatty acid composition to different extents, and that strong interactions between these factors exist. The most aberrant lipid profiles were observed in APP/PS1 animals receiving DHA-poor diets and deprived of estrogens. Conversely, wild-type animals under a high-DHA diet and receiving estradiol exhibited a lipid profile that closely resembled that of the hippocampus of control animals. Interestingly, though the lipid signatures of APP/PS1 hippocampi markedly differed from wild-type, administration of a high-DHA diet in the presence of estrogens gave rise to a lipid profile that approached that of control animals. Paralleling changes in lipid composition, patterns of gene expression of enzymes involved in lipid biosynthesis were also altered and affected by combination of experimental factors. Overall, these results indicate that hippocampal lipid homeostasis is strongly affected by hormonal and dietary conditions, and that manipulation of these factors might be incorporated in AD therapeutics.

Cellular and Molecular Basis for Acute Nongenomically Mediated Actions of SERMs

Compelling evidence accumulated over the past three decades have demonstrated that, besides their ability to antagonize estrogen binding to their intracellular specific estrogen receptors (ER), selective estrogen receptor modulators (SERMs) can affect a number of biochemical processes in eukaryotic cells. Experimental data from in vivo and in vitro studies have revealed that SERMs and estrogens are surprisingly pleiotropic molecules affecting molecular targets in both estrogen receptor positive (ER+) and negative (ER–) cells. Such “alternative” actions of SERMs and estrogens are typically independent of canonical ERs and do not involve transcriptional or translational events, thereby mediated nongenomically, and usually initiated (and accomplished) within seconds to minutes after presentation of the molecule (Falkestein et al. 2000; Nadal el al. 2001). The spectrumof SERM-induced acute actions includes a wide set of molecular targets, frommodulation of ion channels and signaling molecules to alteration of membrane fluidity. In the following sections we review data from different laboratories, including ours, in the context of cellular and molecular evidences for acute nongenomic effects of SERMs observed at pharmacological circulating concentrations. Special emphasis will be placed on actions that might underlie clinically relevant beneficial effects as well as undesirable side effects.

Effects of vasopressin on electrolyte transport in lizard intestine

SummaryVasopressin applied serosally had no effect on electrical parameters and unidirectional Na and Cl fluxes across anin vitro short circuited preparation of lizard ileum. Short circuit current (Isc) and transmural potential difference (PD) across colon were decreased by vasopressin and increased by cyclic AMP. Vasopressin increased the mucosal-to-serosal flux of sodium and chloride across short circuited colon. Cyclic AMP had no effect on the rate of Na absorption but reversed Cl absorption to secretion. Vasopressin enhanced the net absorption of water across the colon but had no effect on absorption across ileum. Cyclic AMP activity in homogenates of colon was not altered by vasopressin but was increased by theophylline. It is concluded that the colonic response of the lizard colon to vasopressin is mediated by a noncyclic AMP mechanism.

Polyamines transduce the nongenomic, androgen-induced calcium sensitization in intestinal smooth muscle.

Androgens regulate body development and differentiation through a variety of genotropic mechanisms, mostly in reproductive organs. In recent years a different scenario for sex hormone actions has emerged: the intestinal muscle. Thus, although estrogens relax intestinal muscle, androgens are powerful inducers of mechanical potentiation. This effect of androgens was intriguing because it is observed at physiological concentrations, is mediated by nongenomic mechanisms, and involves a phenomenon of calcium sensitization of contractile machinery by stimulating phosphorylation of 20 kDa myosin light chain by Rho-associated kinase. Here we have deciphered the molecular mechanisms underlying calcium sensitization and mechanical potentiation by androgens in male intestinal muscle as well as its tight relationship to polyamine metabolism. Thus, androgens stimulate polyamine synthesis, and the inhibition of polyamine synthesis abolishes androgen-induced calcium sensitization and 20 kDa myosin light chain phosphorylation. We demonstrate that the first molecular step in the induction of calcium sensitization is a nonconventional activation of the adaptor protein RhoA, triggered by a transglutaminase-catalyzed polyamination of RhoA, which is then targeted to the membrane to activate Rho-associated kinase. Altogether, these results demonstrate that the physiological levels of androgens, through the modulation of polyamine metabolism and posttanslational modification of RhoA, activate a new signal transduction pathway in the intestinal smooth muscle to induce calcium sensitization. Furthermore, apart from being one of the few physiologically relevant nongenomic effects of androgens, these results might underlie the well-known gender differences in intestinal transits, thus expanding the natures inventory of sex hormones effects.