Testosterone Enhances KV Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y4 Receptors and Adenylyl Cyclase Pathway.

Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.

Chamaecyparis lawsoniana and Its Active Compound Quercetin as Ca2+ Inhibitors in the Contraction of Airway Smooth Muscle.

The Cupressaceae family includes species considered to be medicinal. Their essential oil is used for headaches, colds, cough, and bronchitis. Cedar trees like Chamaecyparis lawsoniana (C. lawsoniana) are commonly found in urban areas. We investigated whether C. lawsoniana exerts some of its effects by modifying airway smooth muscle (ASM) contractility. The leaves of C. lawsoniana (363 g) were pulverized mechanically, and extracts were obtained by successive maceration 1:10 (w:w) with methanol/CHCl3. Guinea pig tracheal rings were contracted with KCl, tetraethylammonium (TEA), histamine (HIS), or carbachol (Cch) in organ baths. In the Cch experiments, tissues were pre-incubated with D-600, an antagonist of L-type voltage-dependent Ca2+ channels (L-VDCC) before the addition of C. lawsoniana. Interestingly, at different concentrations, C. lawsoniana diminished the tracheal contractions induced by KCl, TEA, HIS, and Cch. In ASM cells, C. lawsoniana significantly diminished L-type Ca2+ currents. ASM cells stimulated with Cch produced a transient Ca2+ peak followed by a sustained plateau maintained by L-VDCC and store-operated Ca2+ channels (SOCC). C. lawsoniana almost abolished this last response. These results show that C. lawsoniana, and its active metabolite quercetin, relax the ASM by inhibiting the L-VDCC and SOCC; further studies must be performed to obtain the complete set of metabolites of the extract and study at length their pharmacological properties.

Reconstruction of Anophthalmic Sockets With Pedicled Nasoseptal Flap.

PURPOSE: Patients who undergo eye removal often present with orbital soft-tissue insufficiency and contraction of the eye sockets. The most commonly used reconstruction strategy is grafting the orbit with free grafts, which is associated with the drawback of harvesting tissue from an unconnected site. This study describes the use of the vascularized nasoseptal flap in the reconstruction and enlargement of the contracted anophthalmic cavity in patients with severe or recurrent contracted eye sockets and evaluates its efficacy. METHODS: A sphenopalatine-pedicled flap from the nasal septum was harvested and mobilized into the anophthalmic orbit for the reconstruction, coverage, and enlargement of the socket in 17 patients with anophthalmic socket syndrome. Data regarding the demographics, preoperative status, postoperative findings, follow-up, outcomes, dates of mutilant and reconstructive surgery, and relevant clinical or imaging were collected. RESULTS: Krishna´s classification was used to assess the postoperative outcomes. The final rating improved in all patients at a median follow-up duration of 35 months. A greater impact was observed in patients who underwent reconstructive surgery before nasoseptal flap creation. Two minor complications occurred; however, major surgical intervention was not required. Implant extrusion was observed in 2 patients. CONCLUSIONS: The novel strategy of applying nasoseptal flaps in the reconstruction of anophthalmic sockets results in improved socket grading and a low rate of recurrence (socket contracture or implant extrusion), and complications. The vascular nature of the flap makes it suitable for use in complex cases.

Binding of YY1/CREB to an Enhancer Region Triggers Claudin 6 Expression in H. pylori LPS-Stimulated AGS Cells.

Aberrant expression of the tight junction protein claudin 6 (CLDN6) is a hallmark of gastric cancer progression. Its expression is regulated by the cAMP response element-binding protein (CREB). In gastric cancer induced by Helicobacter pylori (H. pylori) there is no information regarding what transcription factors induce/upregulate the expression of CLDN6. We aimed to identify whether CREB and Yin Yang1 (YY1) regulate the expression of CLDN6 and the site where they bind to the promoter sequence. Bioinformatics analysis, H. pylori lipopolysaccharide (LPS), YY1 and CREB silencing, Western blot, luciferase assays, and chromatin immunoprecipitation experiments were performed using the stomach gastric adenocarcinoma cell line AGS. A gen reporter assay suggested that the initial 2000 bp contains the regulatory sequence associated with CLDN6 transcription; the luciferase assay demonstrated three different regions with transcriptional activity, but the -901 to -1421 bp region displayed the maximal transcriptional activity in response to LPS. Fragment 1279-1421 showed CREB and, surprisingly, YY1 occupancy. Sequential Chromatin Immunoprecipitation (ChIP) experiments confirmed that YY1 and CREB interact in the 1279-1421 region. Our results suggest that CLDN6 expression is regulated by the binding of YY1 and CREB in the 901-1421 enhancer, in which a non-described interaction of YY1 with CREB was established in the 1279-1421 region.

Theophylline-Induced Relaxation Is Enhanced after Testosterone Treatment via Increased KV1.2 and KV1.5 Protein Expression in Guinea Pig Tracheal Smooth Muscle.

Theophylline is a drug commonly used to treat asthma due to its anti-inflammatory and bronchodilatory properties. Testosterone (TES) has been suggested to reduce the severity of asthma symptoms. This condition affects boys more than girls in childhood, and this ratio reverses at puberty. We reported that guinea pig tracheal tissue chronic exposure to TES increases the expression of ß2-adrenoreceptors and enhances salbutamol-induced K+ currents (IK+). Herein, we investigated whether the upregulation of K+ channels can enhance the relaxation response to methylxanthines, including theophylline. Chronic incubation of guinea pig tracheas with TES (40 nM, 48 h) enhanced the relaxation induced by caffeine, isobutylmethylxanthine, and theophylline, an effect that was abolished by tetraethylammonium. In tracheal myocytes, chronic incubation with TES increased theophylline-induced IK+; flutamide reversed this effect. The increase in IK+ was blocked by 4-aminopyridine by ~82%, whereas iberiotoxin reduced IK+ by ~17%. Immunofluorescence studies showed that chronic TES exposure increased the expression of KV1.2 and KV1.5 in airway smooth muscle (ASM). In conclusion, chronic exposure to TES in guinea pig ASM promotes upregulation of KV1.2 and KV1.5 and enhances theophylline relaxation response. Therefore, gender should be considered when prescribing methylxanthines, as teenage boys and males are likely to respond better than females.

Phytoestrogen-Based Hormonal Replacement Therapy Could Benefit Women Suffering Late-Onset Asthma.

It has been observed that plasmatic concentrations of estrogens, progesterone, or both correlate with symptoms in asthmatic women. Fluctuations in female sex steroid concentrations during menstrual periods are closely related to asthma symptoms, while menopause induces severe physiological changes that might require hormonal replacement therapy (HRT), that could influence asthma symptoms in these women. Late-onset asthma (LOA) has been categorized as a specific asthmatic phenotype that includes menopausal women and novel research regarding therapeutic alternatives that might provide relief to asthmatic women suffering LOA warrants more thorough and comprehensive analysis. Therefore, the present review proposes phytoestrogens as a promising HRT that might provide these females with relief for both their menopause and asthma symptoms. Besides their well-recognized anti-inflammatory and antioxidant capacities, phytoestrogens activate estrogen receptors and promote mild hormone-like responses that benefit postmenopausal women, particularly asthmatics, constituting therefore a very attractive potential therapy largely due to their low toxicity and scarce side effects.

Estrogenic Modulation of Ionic Channels, Pumps and Exchangers in Airway Smooth Muscle.

To preserve ionic homeostasis (primarily Ca2+, K+, Na+, and Cl-), in the airway smooth muscle (ASM) numerous transporters (channels, exchangers, and pumps) regulate the influx and efflux of these ions. Many of intracellular processes depend on continuous ionic permeation, including exocytosis, contraction, metabolism, transcription, fecundation, proliferation, and apoptosis. These mechanisms are precisely regulated, for instance, through hormonal activity. The lipophilic nature of steroidal hormones allows their free transit into the cell where, in most cases, they occupy their cognate receptor to generate genomic actions. In the sense, estrogens can stimulate development, proliferation, migration, and survival of target cells, including in lung physiology. Non-genomic actions on the other hand do not imply estrogen's intracellular receptor occupation, nor do they initiate transcription and are mostly immediate to the stimulus. Among estrogen's non genomic responses regulation of calcium homeostasis and contraction and relaxation processes play paramount roles in ASM. On the other hand, disruption of calcium homeostasis has been closely associated with some ASM pathological mechanism. Thus, this paper intends to summarize the effects of estrogen on ionic handling proteins in ASM. The considerable diversity, range and power of estrogens regulates ionic homeostasis through genomic and non-genomic mechanisms.

Claudin-6 increases SNAI1, NANOG and SOX2 gene expression in human gastric adenocarcinoma AGS cells.

BACKGROUND: Gastric cancer is a heterogeneous disease associated to deregulated gastric epithelia tight junction barrier function and di novo expression of claudin-6; these changes are associated with epithelial-mesenchymal transition, enhanced invasiveness, metastatic progression, resistance to chemotherapy, and poor prognosis. Gastric cancer stem cells represent a rare population of cells within the tumor implicated in tumor growth and higher tumorigenic capacity. The possible relation between claudin-6 expression and the expression of some markers associated to epithelial mesenchymal transition and cancer stem cells in gastric cancer cells have never been explored. METHODS AND RESULTS: CD44, CD24, Twist, Villin, DCLK1, claudin-6, NANOG, E-Cadherin, SOX2, and SNAI1 expression was evaluated by immunofluorescence and cytofluorometry in wild type and Claudin-6 transfected AGS cells. Cell migration assays were also performed. Differentially expressed genes and biological processes analysis was performed to determine gene preponderance. The results showed that claudin-6 overexpression enriched the CD44 + /CD24- subpopulation with an overall increase in the expression and the number of CD44 + cells. A significant increase in NANOG, SOX2 and SNAI1 expression and enhanced cell migration was observed in claudin-6 transfected cells. Transcriptome analysis revealed 271 genes involved in enhanced biological processes with only 31 with a significantly p value; thirteen of those genes are closely associated to epithelial mesenchymal transition processes and folding and unfolding processes of proteins in the endoplasmic reticulum. CONCLUSIONS: The pro-tumorigenic effect of claudin-6 in gastric cancer could be associated to dedifferentiation of epithelial cells and an increase in di novo cancer stem cell genesis.

RL-DOVS: Reinforcement Learning for Autonomous Robot Navigation in Dynamic Environments.

Autonomous navigation in dynamic environments where people move unpredictably is an essential task for service robots in real-world populated scenarios. Recent works in reinforcement learning (RL) have been applied to autonomous vehicle driving and to navigation around pedestrians. In this paper, we present a novel planner (reinforcement learning dynamic object velocity space, RL-DOVS) based on an RL technique for dynamic environments. The method explicitly considers the robot kinodynamic constraints for selecting the actions in every control period. The main contribution of our work is to use an environment model where the dynamism is represented in the robocentric velocity space as input to the learning system. The use of this dynamic information speeds the training process with respect to other techniques that learn directly either from raw sensors (vision, lidar) or from basic information about obstacle location and kinematics. We propose two approaches using RL and dynamic obstacle velocity (DOVS), RL-DOVS-A, which automatically learns the actions having the maximum utility, and RL-DOVS-D, in which the actions are selected by a human driver. Simulation results and evaluation are presented using different numbers of active agents and static and moving passive agents with random motion directions and velocities in many different scenarios. The performance of the technique is compared with other state-of-the-art techniques for solving navigation problems in environments such as ours.

Robot Localization in Tunnels: Combining Discrete Features in a Pose Graph Framework.

Robot localization inside tunnels is a challenging task due to the special conditions of these environments. The GPS-denied nature of these scenarios, coupled with the low visibility, slippery and irregular surfaces, and lack of distinguishable visual and structural features, make traditional robotics methods based on cameras, lasers, or wheel encoders unreliable. Fortunately, tunnels provide other types of valuable information that can be used for localization purposes. On the one hand, radio frequency signal propagation in these types of scenarios shows a predictable periodic structure (periodic fadings) under certain settings, and on the other hand, tunnels present structural characteristics (e.g., galleries, emergency shelters) that must comply with safety regulations. The solution presented in this paper consists of detecting both types of features to be introduced as discrete sources of information in an alternative graph-based localization approach. The results obtained from experiments conducted in a real tunnel demonstrate the validity and suitability of the proposed system for inspection applications.

Chromosomally Unstable Gastric Cancers Overexpressing Claudin-6 Disclose Cross-Talk between HNF1A and HNF4A, and Upregulated Cholesterol Metabolism.

(1) Abnormally increased expression of claudin-6 in gastric cancer is considered a prognostic marker of the chromosomal unstable molecular subtype. However, a detailed molecular profile analysis of differentially expressed genes and affected pathways associated with claudin-6 increased (Cldn6high) expression has not been assessed. (2) The TCGA Stomach Adenocarcinoma Pan-Cancer Atlas Data was evaluated using Cytoscape's Gene Mania, MCODE, and Cytohubba bioinformatic software. (3) 96.88% of Cldn6high gastric cancer tumors belonging to the chromosomal unstable molecular subtype are associated with a worse prognosis. Cldn6expression coincided with higher mutations in TP53, MIEN1, STARD3, PGAP3, and CCNE1 genes compared to Cldn6low expression. In Cldn6high cancers, 1316 genes were highly expressed. Cholesterol metabolism was the most affected pathway as APOA1, APOA2, APOH, APOC2, APOC3, APOB-100, LDL receptor-related protein 1/2, Sterol O-acyltransferase, STARD3, MAGEA-2, -3, -4, -6, -9B, and -12 genes were overexpressed in Cldn6high gastric cancers; interestingly, APOA2 and MAGEA9b were identified as top hub genes. Functional enrichment of DEGs linked HNF-4α and HNF-1α genes as highly expressed in Cldn6high gastric cancer. (4) Our results suggest that APOA2 and MAGEA9b could be considered as prognostic markers for Cldn6high gastric cancers.

Could Lower Testosterone in Older Men Explain Higher COVID-19 Morbidity and Mortalities?

The health scourge imposed on humanity by the COVID-19 pandemic seems not to recede. This fact warrants refined and novel ideas analyzing different aspects of the illness. One such aspect is related to the observation that most COVID-19 casualties were older males, a tendency also noticed in the epidemics of SARS-CoV in 2003 and the Middle East respiratory syndrome in 2012. This gender-related difference in the COVID-19 death toll might be directly involved with testosterone (TEST) and its plasmatic concentration in men. TEST has been demonstrated to provide men with anti-inflammatory and immunological advantages. As the plasmatic concentration of this androgen decreases with age, the health benefit it confers also diminishes. Low plasmatic levels of TEST can be determinant in the infection's outcome and might be related to a dysfunctional cell Ca2+ homeostasis. Not only does TEST modulate the activity of diverse proteins that regulate cellular calcium concentrations, but these proteins have also been proven to be necessary for the replication of many viruses. Therefore, we discuss herein how TEST regulates different Ca2+-handling proteins in healthy tissues and propose how low TEST concentrations might facilitate the replication of the SARS-CoV-2 virus through the lack of modulation of the mechanisms that regulate intracellular Ca2+ concentrations.

Theophylline: Old Drug in a New Light, Application in COVID-19 through Computational Studies.

Theophylline (3-methyxanthine) is a historically prominent drug used to treat respiratory diseases, alone or in combination with other drugs. The rapid onset of the COVID-19 pandemic urged the development of effective pharmacological treatments to directly attack the development of new variants of the SARS-CoV-2 virus and possess a therapeutical battery of compounds that could improve the current management of the disease worldwide. In this context, theophylline, through bronchodilatory, immunomodulatory, and potentially antiviral mechanisms, is an interesting proposal as an adjuvant in the treatment of COVID-19 patients. Nevertheless, it is essential to understand how this compound could behave against such a disease, not only at a pharmacodynamic but also at a pharmacokinetic level. In this sense, the quickest approach in drug discovery is through different computational methods, either from network pharmacology or from quantitative systems pharmacology approaches. In the present review, we explore the possibility of using theophylline in the treatment of COVID-19 patients since it seems to be a relevant candidate by aiming at several immunological targets involved in the pathophysiology of the disease. Theophylline down-regulates the inflammatory processes activated by SARS-CoV-2 through various mechanisms, and herein, they are discussed by reviewing computational simulation studies and their different applications and effects.

Sex Hormones and Lung Inflammation.

Inflammation is a characteristic marker in numerous lung disorders. Several immune cells, such as macrophages, dendritic cells, eosinophils, as well as T and B lymphocytes, synthetize and release cytokines involved in the inflammatory process. Gender differences in the incidence and severity of inflammatory lung ailments including asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (PF), lung cancer (LC), and infectious related illnesses have been reported. Moreover, the effects of sex hormones on both androgens and estrogens, such as testosterone (TES) and 17ß-estradiol (E2), driving characteristic inflammatory patterns in those lung inflammatory diseases have been investigated. In general, androgens seem to display anti-inflammatory actions, whereas estrogens produce pro-inflammatory effects. For instance, androgens regulate negatively inflammation in asthma by targeting type 2 innate lymphoid cells (ILC2s) and T-helper (Th)-2 cells to attenuate interleukin (IL)-17A-mediated responses and leukotriene (LT) biosynthesis pathway. Estrogens may promote neutrophilic inflammation in subjects with asthma and COPD. Moreover, the activation of estrogen receptors might induce tumorigenesis. In this chapter, we summarize the most recent advances in the functional roles and associated signaling pathways of inflammatory cellular responses in asthma, COPD, PF, LC, and newly occurring COVID-19 disease. We also meticulously deliberate the influence of sex steroids on the development and progress of these common and severe lung diseases.

Possible Beneficial Actions of Caffeine in SARS-CoV-2.

The COVID-19 pandemic has established an unparalleled necessity to rapidly find effective treatments for the illness; unfortunately, no specific treatment has been found yet. As this is a new emerging chaotic situation, already existing drugs have been suggested to ameliorate the infection of SARS-CoV-2. The consumption of caffeine has been suggested primarily because it improves exercise performance, reduces fatigue, and increases wakefulness and awareness. Caffeine has been proven to be an effective anti-inflammatory and immunomodulator. In airway smooth muscle, it has bronchodilator effects mainly due to its activity as a phosphodiesterase inhibitor and adenosine receptor antagonist. In addition, a recent published document has suggested the potential antiviral activity of this drug using in silico molecular dynamics and molecular docking; in this regard, caffeine might block the viral entrance into host cells by inhibiting the formation of a receptor-binding domain and the angiotensin-converting enzyme complex and, additionally, might reduce viral replication by the inhibition of the activity of 3-chymotrypsin-like proteases. Here, we discuss how caffeine through certain mechanisms of action could be beneficial in SARS-CoV-2. Nevertheless, further studies are required for validation through in vitro and in vivo models.

Vanadium compounds and cellular death mechanisms in the A549 cell line: The relevance of the compound valence.

Non-small lung cell carcinoma has a high morbidity and mortality rates. The elective treatment for stage III and IV is cisplatinum that conveys serious toxic side effects. Vanadium compounds are metal molecules with proven antitumor activity that depends on its valence. Therefore, a better understanding of the mechanism of action of vanadium compounds is required. The aim of our study was to investigate the mechanisms of cell death induced by sodium metavanadate (NaVO3 [V(+5)]) and vanadyl sulfate (VOSO4 [(+4)]), both of which have reported apoptotic-inducing activity. We exposed the A549 cell line to various concentrations (0-100 µM) and to different exposure times to each compound and determined the cell viability and expression of caspases, reactive oxygen species (ROS) production, Bcl2, Bax, FasL and NO. Our results showed that neither compounds modified the basal expression of caspases or pro- and anti-apoptotic proteins. The only change observed was the 12- and 14-fold significant increase in ROS production induced by NaVO3 and VOSO4 , respectively, at 100 µm concentrations after 48 hours. Our results suggest that classical apoptotic mechanisms are not related to the cell death induced by the vanadium compounds evaluated here, and showed that the higher ROS production was induced by the [(+4)] valence compound. It is possible that the difference will be secondary to its higher oxidative status and thus higher ROS production, which leads to higher cell damage. In conclusion, our results suggest that the efficacy of the cell death mechanisms induced by vanadium compounds differ depending on the valence of the compound.

17ß-Estradiol modulates cell proliferation of medullary cords during ovarian differentiation of the Lepidochelys olivacea sea turtle.

In turtles undergoing temperature sex determination (TSD), bipotential gonads express Sox9 in medullary cords at both female- (FPT) and male-producing temperatures (MPT). Subsequently, when the sex fate of medullary cords becomes dimorphic, at FPT, Sox9 is downregulated, whereas at MPT, its expression is maintained. Medullary cords in the ovary turn into ovarian lacuna, whereas in the testis they differentiate as seminiferous cords. When embryos of Lepidochelys olivacea sea turtle are incubated at MPT and treated with estradiol, Sox9 expression persists in the medullary cords in the form of tiny ovotestis-like formations. The perturbed development of the treated gonads is due to a significant decrease in the number of proliferating cells. This suggests that the disturbed effect caused by exogenous estradiol may be due to a conflict between the gene networks regulated by temperature and the increased level of endogenous estrogens, induced by the treatment. Here, we decided to use fadrozole and fulvestrant, an aromatase inhibitor and an estrogen-receptor antagonist, respectively, to provide insights into the role played by endogenous estrogens in regulating the cell proliferation of the two main gonadal compartments: the medullary cords and the cortex. Comparing cell proliferation patterns, our current results suggest that the endogenous estrogens are involved in determining the sex fate of medullary cords, by repressing proliferation. Interestingly, our results showed that endogenous estradiol levels are unnecessary for the thickening of the ovarian cortex.

The aminoestrogen prolame increases recognition memory and hippocampal neuronal spine density in aged mice.

The aging brain shows biochemical and morphological changes in the dendrites of pyramidal neurons from the limbic system associated with memory loss. Prolame (N-(3-hydroxy-1,3,5 (10)-estratrien-17ß-yl)-3-hydroxypropylamine) is a non-feminizing aminoestrogen with antithrombotic activity that prevents neuronal deterioration, oxidative stress, and neuroinflammation. Our aim was to evaluate the effect of prolame on motor and cognitive processes, as well as its influence on the dendritic morphology of neurons at the CA1, CA3, and granule cells of the dentate gyrus (DG) regions of hippocampus (HP), and medium spiny neurons of the nucleus accumbens (NAcc) of aged mice. Dendritic morphology was assessed with the Golgi-Cox stain procedure followed by Sholl analysis. Prolame (60 µg/kg) was subcutaneously injected daily for 60 days in 18-month-old mice. Immediately after treatment, locomotor activity in a new environment and recognition memory using the Novel Object Recognition Task (NORT) were evaluated. Prolame-treated mice showed a significant increase in the long-term exploration quotient, but locomotor activity was not modified in comparison to control animals. Prolame-treated mice showed a significant increase in dendritic spines density and dendritic length in neurons of the CA1, CA3, and DG regions of the HP, whereas dendrites of neurons in the NAcc remained unmodified. In conclusion, prolame administration promotes hippocampal plasticity processes but not in the NAcc neurons of aged mice, thus improving long-term recognition memory. Prolame could become a pharmacological alternative to prevent or delay the brain aging process, and thus the emergence of neurodegenerative diseases that affect memory.

Heteromeric channels with different phenotypes are generated when coexpressing two P2X2 receptor isoforms.

To investigate if channels with different stoichiometry are formed from P2X2 receptor isoforms during their heterologous co-expression. The two-electrode voltage-clamp technique was used to measured ATP induced currents in Xenopus laevis oocytes. We used a mutant (P2X2-2bm) because its ATP sensitivity is lower than P2X2-2b receptors, which highlights the differences with its splice variant P2X2-1a.Currents through homomeric channels had significantly different Hill coefficients. P2XR are trimeric proteins with three agonist binding sites; therefore, only two homomeric and two heteromeric stoichiometries are possible when both P2X2 isoforms are coexpressed, the heteromeric channels might be formed by: i) 2(P2X2-1a)+1(P2X2-2bm); or ii) 1(P2X2-1a)+2(P2X2-2bm). Because P2X2 channels open when two binding sites are occupied, these stoichiometries are expected to have different ATP sensitivities. Thus, co-expressing both P2X2 isoforms, two oocyte populations were distinguished based on their sensitivity to ATP and Hill coefficients. For the first population (P2X2-1a like), the ATP EC50 and the Hill coefficient were not different than those of homomeric P2X2-1a channels similarly, for the second population (P2X2-2bm like), these variables were also not different than for those of homomeric P2X2-2bm channels. Various findings indicate that homomeric channel expression is not responsible for such differences. Our observations indicate that two heteromeric channels can be assembled from two P2X2 receptor isoforms. Our data support a current model, according to which, ATP activation of two subunits can open P2X2 channel. However, PPADS appears to bind to all three subunits in order to inhibit ATP effects on P2X2 receptors.

Tumor Necrosis Factor Alpha Inhibits L-Type Ca(2+) Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway.

Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca(2+) channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway.