RESUMO
BACKGROUND: Vascular large conductance Ca2+-activated K+ (BK) channel, composed of the α-subunit (BK-α) and the ß1-subunit (BK-ß1), is a key determinant of coronary vasorelaxation and its function is impaired in diabetic vessels. However, our knowledge of diabetic BK channel dysregulation is incomplete. The Sorbs2 (Sorbin homology [SoHo] and Src homology 3 [SH3] domains-containing protein 2), is ubiquitously expressed in arteries, but its role in vascular pathophysiology is unknown. METHODS: The role of Sorbs2 in regulating vascular BK channel activity was determined using patch-clamp recordings, molecular biological techniques, and in silico analysis. RESULTS: Sorbs2 is not only a cytoskeletal protein but also an RNA-binding protein that binds to BK channel proteins and BK-α mRNA, regulating BK channel expression and function in coronary smooth muscle cells. Molecular biological studies reveal that the SH3 domain of Sorbs2 is necessary for Sorbs2 interaction with BK-α subunits, while both the SH3 and SoHo domains of Sorbs2 interact with BK-ß1 subunits. Deletion of the SH3 or SoHo domains abolishes the Sorbs2 effect on the BK-α/BK-ß1 channel current density. Additionally, Sorbs2 is a target gene of the Nrf2 (nuclear factor erythroid-2-related factor 2), which binds to the promoter of Sorbs2 and regulates Sorbs2 expression in coronary smooth muscle cells. In vivo studies demonstrate that Sorbs2 knockout mice at 4 months of age display a significant decrease in BK channel expression and function, accompanied by impaired BK channel Ca2+-sensitivity and BK channel-mediated vasodilation in coronary arteries, without altering their body weights and blood glucose levels. Importantly, Sorbs2 expression is significantly downregulated in the coronary arteries of db/db type 2 diabetic mice. CONCLUSIONS: Sorbs2, a downstream target of Nrf2, plays an important role in regulating BK channel expression and function in vascular smooth muscle cells. Vascular Sorbs2 is downregulated in diabetes. Genetic knockout of Sorbs2 manifests coronary BK channelopathy and vasculopathy observed in diabetic mice, independent of obesity and glucotoxicity.
Assuntos
Canalopatias , Diabetes Mellitus Experimental , Camundongos , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Canalopatias/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Vasos Coronários/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Progesterone (≥1 µM) is used in recovery of cerebral ischemia, an effect likely contributed to by cerebrovascular dilation. The targets of this progesterone action are unknown. We report that micromolar (µM) progesterone activates mouse cerebrovascular myocyte BK channels; this action is lost in ß1-/- mice myocytes and in lipid bilayers containing BK α subunit homomeric channels but sustained on ß1/ß4-containing heteromers. Progesterone binds to both regulatory subunits, involving two steroid binding sites conserved in ß1-ß4: high-affinity (sub-µM), which involves Trp87 in ß1 loop, and low-affinity (µM) defined by TM1 Tyr32 and TM2 Trp163. Thus progesterone, but not its oxime, bridges TM1-TM2. Mutation of the high-affinity site blunts channel activation by progesterone underscoring a permissive role of the high-affinity site: progesterone binding to this site enables steroid binding at the low-affinity site, which activates the channel. In support of our model, cerebrovascular dilation evoked by µM progesterone is lost by mutating Tyr32 or Trp163 in ß1 whereas these mutations do not affect alcohol-induced cerebrovascular constriction. Furthermore, this alcohol action is effectively counteracted both in vitro and in vivo by progesterone but not by its oxime.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta , Progesterona , Camundongos , Animais , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Progesterona/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Esteroides/farmacologia , Sítios de Ligação , Etanol/farmacologia , Oximas/farmacologiaAssuntos
Neoplasias Ovarianas , RNA Longo não Codificante , Feminino , Humanos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Oncogenes/genética , Neoplasias Ovarianas/genética , RNA Longo não Codificante/genéticaRESUMO
Calcium/voltage-activated potassium channels (BK) control smooth muscle (SM) tone and cerebral artery diameter. They include channel-forming α and regulatory ß1 subunits, the latter being highly expressed in SM. Both subunits participate in steroid-induced modification of BK activity: ß1 provides recognition for estradiol and cholanes, resulting in BK potentiation, whereas α suffices for BK inhibition by cholesterol or pregnenolone. Aldosterone can modify cerebral artery function independently of its effects outside the brain, yet BK involvement in aldosterone's cerebrovascular action and identification of channel subunits, possibly involved in steroid action, remains uninvestigated. Using microscale thermophoresis, we demonstrated that each subunit type presents two recognition sites for aldosterone: at 0.3 and ≥10 µM for α and at 0.3-1 µM and ≥100 µM for ß1. Next, we probed aldosterone on SM BK activity and diameter of middle cerebral artery (MCA) isolated from ß1-/- vs. wt mice. Data showed that ß1 leftward-shifted aldosterone-induced BK activation, rendering EC50~3 µM and ECMAX ≥ 10 µM, at which BK activity increased by 20%. At similar concentrations, aldosterone mildly yet significantly dilated MCA independently of circulating and endothelial factors. Lastly, aldosterone-induced MCA dilation was lost in ß1-/- mice. Therefore, ß1 enables BK activation and MCA dilation by low µM aldosterone.
Assuntos
Aldosterona , Canais de Potássio Ativados por Cálcio de Condutância Alta , Camundongos , Animais , Aldosterona/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Músculo Liso Vascular , Dilatação , Esteroides/farmacologia , Artérias CerebraisRESUMO
Alcohol intake leading to blood ethanol concentrations (BEC) ≥ legal intoxication modifies brain blood flow with increases in some regions and decreases in others. Brain regions receive blood from the Willis' circle branches: anterior, middle (MCA) and posterior cerebral (PCA), and basilar (BA) arteries. Rats and mice have been used to identify the targets mediating ethanol-induced effects on cerebral arteries, with conclusions being freely interchanged, albeit data were obtained in different species/arterial branches. We tested whether ethanol action on cerebral arteries differed between male rat and mouse and/or across different brain regions and identified the targets of alcohol action. In both species and all Willis' circle branches, ethanol evoked reversible and concentration-dependent constriction (EC50s ≈ 37-86 mM; below lethal BEC in alcohol-naïve humans). Although showing similar constriction to depolarization, both species displayed differential responses to ethanol: in mice, MCA constriction was highly sensitive to the presence/absence of the endothelium, whereas in rat PCA was significantly more sensitive to ethanol than its mouse counterpart. In the rat, but not the mouse, BA was more ethanol sensitive than other branches. Both interspecies and regional variability were ameliorated by endothelium. Selective large conductance (BK) channel block in de-endothelialized vessels demonstrated that these channels were the effectors of alcohol-induced cerebral artery constriction across regions and species. Variabilities in alcohol actions did not fully matched KCNMB1 expression across vessels. However, immunofluorescence data from KCNMB1-/- mouse arteries electroporated with KCNMB1-coding cDNA demonstrate that KCNMB1 proteins, which regulate smooth muscle (SM) BK channel function and vasodilation, regulate interspecies and regional variability of brain artery responses to alcohol.
Assuntos
Artérias Cerebrais , Etanol , Animais , Masculino , Camundongos , Ratos , Etanol/farmacologia , Etanol/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/metabolismo , Ratos Sprague-DawleyRESUMO
Significant progress has been made in elucidating the basic principles that govern neuronal specification in the developing central nervous system. In contrast, much less is known about the origin of astrocytic diversity. Here, we demonstrate that a restricted pool of progenitors in the mouse spinal cord, expressing the transcription factor Dbx1, produces a subset of astrocytes, in addition to interneurons. Ventral p0-derived astrocytes (vA0 cells) exclusively populate intermediate regions of spinal cord with extraordinary precision. The postnatal vA0 population comprises gray matter protoplasmic and white matter fibrous astrocytes and a group of cells with strict radial morphology contacting the pia. We identified that vA0 cells in the lateral funiculus are distinguished by the expression of reelin and Kcnmb4. We show that Dbx1 mutants have an increased number of vA0 cells at the expense of p0-derived interneurons. Manipulation of the Notch pathway, together with the alteration in their ligands seen in Dbx1 knockouts, suggest that Dbx1 controls neuron-glial balance by modulating Notch-dependent cell interactions. In summary, this study highlights that restricted progenitors in the dorsal-ventral neural tube produce region-specific astrocytic subgroups and that progenitor transcriptional programs highly influence glial fate and are instrumental in creating astrocyte diversity.
Assuntos
Astrócitos , Medula Espinal , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Interneurônios/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Camundongos , Neuroglia/metabolismo , Transdução de Sinais/genética , Medula Espinal/metabolismoRESUMO
Large conductance, calcium/voltage-gated potassium channels (BK) regulate critical body processes, including neuronal, secretory and smooth muscle (SM) function. While BK-forming alpha subunits are ubiquitous, accessory beta1 subunits are highly expressed in SM. This makes beta1 an attractive target for pharmaceutical development to treat SM disorders, such as hypertension or cerebrovascular spasm. Compounds activating BK via beta1 have been identified, yet they exhibit low potency and off-target effects while antagonists that limit agonist activity via beta 1 remain unexplored. Beta1-dependent BK ligand-based pharmacophore modeling and ZINC database searches identified 15 commercially available hits. Concentration-response curves on BK alpha + beta1 subunit-mediated currents were obtained in CHO cells. One potent (EC50 = 20 nM) and highly efficacious activator (maximal activation = ×10.3 of control) was identified along with a potent antagonist (KB = 3.02 nM), both of which were dependent on beta1. Our study provides the first proof-of-principle that an agonist/antagonist pair can be used to control beta1-containing BK activity.
Assuntos
Cálcio , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta , Animais , Cálcio/metabolismo , Cricetinae , Cricetulus , Canais de Potássio Ativados por Cálcio de Condutância Alta , Técnicas de Patch-ClampRESUMO
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new member of the Betacoronaviridae family, responsible for the recent pandemic outbreak of COVID-19. To start exploring the molecular events that follow host cell infection, we queried VirusCircBase and identified a circular RNA (circRNA) predicted to be synthesized by SARS-CoV-2, circ_3205, which we used to probe: (i) a training cohort comprised of two pools of cells from three nasopharyngeal swabs of SARS-CoV-2 infected (positive) or uninfected (negative, UCs) individuals; (ii) a validation cohort made up of 12 positive and 3 negative samples. The expression of circRNAs, miRNAs and miRNA targets was assayed through real-time PCR. CircRNA-miRNA interactions were predicted by TarpMiR, Analysis of Common Targets for circular RNAs (ACT), and STarMir tools. Enrichment of the biological processes and the list of predicted miRNA targets were retrieved from DIANA miRPath v3.0. Our results showed that the predicted SARS-CoV-2 circ_3205 was expressed only in positive samples and its amount positively correlated with that of SARS-CoV-2 Spike (S) mRNA and the viral load (r values = 0.80952 and 0.84867, Spearman's correlation test, respectively). Human (hsa) miR-298 was predicted to interact with circ_3205 by all three predictive tools. KCNMB4 and PRKCE were predicted as hsa-miR-298 targets. Interestingly, the function of both is correlated with blood coagulation and immune response. KCNMB4 and PRKCE mRNAs were upregulated in positive samples as compared to UCs (6 and 8.1-fold, p values = 0.049 and 0.02, Student's t test, respectively) and their expression positively correlated with that of circ_3205 (r values = 0.6 and 0.25, Spearman's correlation test, respectively). We propose that our results convincingly suggest that circ_3205 is a circRNA synthesized by SARS-CoV-2 upon host cell infection and that it may behave as a competitive endogenous RNA (ceRNA), sponging hsa-miR-298 and contributing to the upregulation of KCNMB4 and PRKCE mRNAs.
Assuntos
COVID-19/genética , COVID-19/metabolismo , RNA Circular/genética , RNA Viral , SARS-CoV-2/genética , Biologia Computacional , Regulação Viral da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Nasofaringe/virologia , Proteínas do Tecido Nervoso/genética , Mapeamento de Interação de Proteínas , Proteína Quinase C-épsilon/genética , Reprodutibilidade dos TestesRESUMO
The aim of the study was to perform family-based association analysis of PRKCB1, CBLN1 and KCNMB4 gene polymorphisms and autism disorder. We comprised 206 Caucasian children with autistic spectrum disorder (ASD) and their biological parents. In transmission/disequilibrium test we observed that T-allele of the rs198198 polymorphism of the PRKCB1 gene was more often transmitted to affected children in the male subgroup (p = 0.010). Additionally, the T carrier state was significantly associated with hypotonia (p = 0.048). In the female subgroup, the T-allele carriers more often showed more mobile/vital behavior (p = 0.046). In conclusion, our study showed that the rs198198 of the PRKCB1 gene may be associated with ASD in men and with some features characteristic for the disorder.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Proteínas do Tecido Nervoso/genética , Proteína Quinase C beta/genética , Precursores de Proteínas/genética , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Criança , Estudos de Coortes , Feminino , Predisposição Genética para Doença , Humanos , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Polônia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC) is the most prevalent subtype of TDP-43 proteinopathy, affecting up to 1/3rd of aged persons. LATE-NC often co-occurs with hippocampal sclerosis (HS) pathology. It is currently unknown why some individuals with LATE-NC develop HS while others do not, but genetics may play a role. Previous studies found associations between LATE-NC phenotypes and specific genes: TMEM106B, GRN, ABCC9, KCNMB2, and APOE. Data from research participants with genomic and autopsy measures from the National Alzheimer's Coordinating Center (NACC; n = 631 subjects included) and the Religious Orders Study and Memory and the Rush Aging Project (ROSMAP; n = 780 included) were analyzed in the current study. Our goals were to reevaluate disease-associated genetic variants using newly collected data and to query whether the specific genotype/phenotype associations could provide new insights into disease-driving pathways. Research subjects included in prior LATE/HS genome-wide association studies (GWAS) were excluded. Single nucleotide variants (SNVs) within 10 kb of TMEM106B, GRN, ABCC9, KCNMB2, and APOE were tested for association with HS and LATE-NC, and separately for Alzheimer's pathologies, i.e. amyloid plaques and neurofibrillary tangles. Significantly associated SNVs were identified. When results were meta-analyzed, TMEM106B, GRN, and APOE had significant gene-based associations with both LATE and HS, whereas ABCC9 had significant associations with HS only. In a sensitivity analysis limited to LATE-NC + cases, ABCC9 variants were again associated with HS. By contrast, the associations of TMEM106B, GRN, and APOE with HS were attenuated when adjusting for TDP-43 proteinopathy, indicating that these genes may be associated primarily with TDP-43 proteinopathy. None of these genes except APOE appeared to be associated with Alzheimer's-type pathology. In summary, using data not included in prior studies of LATE or HS genomics, we replicated several previously reported gene-based associations and found novel evidence that specific risk alleles can differentially affect LATE-NC and HS.
Assuntos
Apolipoproteínas E/genética , Hipocampo/patologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Progranulinas/genética , Receptores de Sulfonilureias/genética , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Seguimentos , Predisposição Genética para Doença/epidemiologia , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino , Estudos Retrospectivos , EscleroseRESUMO
Calcium-/voltage-gated, large-conductance potassium channels (BKs) control critical physiological processes, including smooth muscle contraction. Numerous observations concur that elevated membrane cholesterol (CLR) inhibits the activity of homomeric BKs consisting of channel-forming alpha subunits. In mammalian smooth muscle, however, native BKs include accessory KCNMB1 (ß1) subunits, which enable BK activation at physiological intracellular calcium. Here, we studied the effect of CLR enrichment on BK currents from rat cerebral artery myocytes. Using inside-out patches from middle cerebral artery (MCA) myocytes at [Ca2+]free=30 µM, we detected BK activation in response to in vivo and in vitro CLR enrichment of myocytes. While a significant increase in myocyte CLR was achieved within 5 min of CLR in vitro loading, this brief CLR enrichment of membrane patches decreased BK currents, indicating that BK activation by CLR requires a protracted cellular process. Indeed, blocking intracellular protein trafficking with brefeldin A (BFA) not only prevented BK activation but led to channel inhibition upon CLR enrichment. Surface protein biotinylation followed by Western blotting showed that BFA blocked the increase in plasmalemmal KCNMB1 levels achieved via CLR enrichment. Moreover, CLR enrichment of arteries with naturally high KCNMB1 levels, such as basilar and coronary arteries, failed to activate BK currents. Finally, CLR enrichment failed to activate BK channels in MCA myocytes from KCNMB1-/- mouse while activation was detected in their wild-type (C57BL/6) counterparts. In conclusion, the switch in CLR regulation of BK from inhibition to activation is determined by a trafficking-dependent increase in membrane levels of KCNMB1 subunits.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Células Musculares/metabolismo , Canais de Potássio/metabolismo , Animais , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Artérias Cerebrais/citologia , Artérias Cerebrais/metabolismo , Colesterol/metabolismo , Colesterol/fisiologia , Vasos Coronários/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Canais de Potássio/fisiologia , Ratos , Ratos Sprague-Dawley , VasoconstriçãoRESUMO
Arachidonic acid (AA) is a fatty acid involved in the modulation of several ion channels. Previously, we reported that AA activates the high conductance Ca2+- and voltage-dependent K+ channel (BK) in vascular smooth muscle depending on the expression of the auxiliary ß1 subunit. Here, using the patch-clamp technique on BK channel co-expressed with ß1 subunit in a heterologous cell expression system, we analyzed whether AA modifies the three functional modules involved in the channel gating: the voltage sensor domain (VSD), the pore domain (PD), and the intracellular calcium sensor domain (CSD). We present evidence that AA activates BK channel in a direct way, inducing VSD stabilization on its active configuration observed as a significant left shift in the Q-V curve obtained from gating currents recordings. Moreover, AA facilitates the channel opening transitions when VSD are at rest, and the CSD are unoccupied. Furthermore, the activation was independent of the intracellular Ca2+ concentration and reduced when the BK channel was co-expressed with the Y74A mutant of the ß1 subunit. These results allow us to present new insigths in the mechanism by which AA modulates BK channels co-expressed with its auxiliary ß1 subunit.
Assuntos
Ácido Araquidônico/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Regulação Alostérica/efeitos dos fármacos , Ácido Araquidônico/química , Células HEK293 , HumanosRESUMO
AIMS: Our recent study demonstrated that increased Ca2+ sparks and spontaneous transient outward currents (STOCs) played an important role in uterine vascular tone and haemodynamic adaptation to pregnancy. The present study examined the role of ryanodine receptor (RyR) subtypes in regulating Ca2+ sparks/STOCs and myogenic tone in uterine arterial adaptation to pregnancy. METHODS AND RESULTS: Uterine arteries isolated from non-pregnant and near-term pregnant sheep were used in the present study. Pregnancy increased the association of α and ß1 subunits of large-conductance Ca2+-activated K+ (BKCa) channels and enhanced the co-localization of RyR1 and RyR2 with the ß1 subunit in the uterine artery. In contrast, RyR3 was not co-localized with BKCa ß1 subunit. Knockdown of RyR1 or RyR2 in uterine arteries of pregnant sheep downregulated the ß1 but not α subunit of the BKCa channel and decreased the association of α and ß1 subunits. Unlike RyR1 and RyR2, knockdown of RyR3 had no significant effect on either expression or association of BKCa subunits. In addition, knockdown of RyR1 or RyR2 significantly decreased Ca2+ spark frequency, suppressed STOCs frequency and amplitude, and increased pressure-dependent myogenic tone in uterine arteries of pregnant animals. RyR3 knockdown did not affect Ca2+ sparks/STOCs and myogenic tone in the uterine artery. CONCLUSION: Together, the present study demonstrates a novel mechanistic paradigm of RyR subtypes in the regulation of Ca2+ sparks/STOCs and uterine vascular tone, providing new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Artéria Uterina/metabolismo , Vasoconstrição , Adaptação Fisiológica , Animais , Pressão Arterial , Feminino , Regulação da Expressão Gênica , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Gravidez , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Carneiro Doméstico , Técnicas de Cultura de TecidosRESUMO
Repetitive transcranial magnetic stimulation (rTMS) is a form of non-invasive brain stimulation frequently used to induce neuroplasticity in the brain. Even at low intensities, rTMS has been shown to modulate aspects of neuronal plasticity such as motor learning and structural reorganisation of neural tissue. However, the impact of low intensity rTMS on glial cells such as astrocytes remains largely unknown. This study investigated changes in RNA (qPCR array: 125 selected genes) and protein levels (immunofluorescence) in cultured mouse astrocytes following a single session of low intensity repetitive magnetic stimulation (LI-rMS - 18 mT). Purified neonatal cortical astrocyte cultures were stimulated with either 1Hz (600 pulses), 10Hz (600 or 6000 pulses) or sham (0 pulses) LI-rMS, followed by RNA extraction at 5 h post-stimulation, or fixation at either 5 or 24-h post-stimulation. LI-rMS resulted in a two-to-four-fold downregulation of mRNA transcripts related to calcium signalling (Stim1 and Orai3), inflammatory molecules (Icam1) and neural plasticity (Ncam1). 10Hz reduced expression of Stim1, Orai3, Kcnmb4, and Ncam1 mRNA, whereas 1Hz reduced expression of Icam1 mRNA and signalling-related genes. Protein levels followed a similar pattern for 10Hz rMS, with a significant reduction of STIM1, ORAI3, KCNMB4, and NCAM1 protein compared to sham, but 1Hz increased STIM1 and ORAI3 protein levels relative to sham. These findings demonstrate the ability of 1Hz and 10Hz LI-rMS to modulate specific aspects of astrocytic phenotype, potentially contributing to the known effects of low intensity rTMS on excitability and neuroplasticity.
Assuntos
Astrócitos , Cálcio , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Inflamação , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta , Fenômenos Magnéticos , Camundongos , Estimulação Magnética TranscranianaRESUMO
Analysis of the presynaptic action potential's (APsyn) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high-resolution optical recordings of membrane potential, exocytosis, and Ca2+ in cultured hippocampal neurons. These recordings revealed a critical and selective role for Kv1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic Kv1 channel inactivation was mediated by the Kvß1 subunit and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of Kvß1 blocked all broadening of the APsyn during high-frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus, using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic Kv channels in synaptic facilitation at presynaptic terminals of the hippocampus upstream of the exocytic machinery.
Assuntos
Hipocampo/metabolismo , Canal de Potássio Kv1.3/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Células Piramidais/metabolismo , Potenciais Sinápticos/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Venenos Elapídicos/farmacologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Microscopia Intravital , Canal de Potássio Kv1.3/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Masculino , Camundongos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Imagem Óptica , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Cultura Primária de Células , Células Piramidais/efeitos dos fármacos , Ratos , Potenciais Sinápticos/efeitos dos fármacosRESUMO
Long noncoding RNA (lncRNA) is emerging as an essential regulator in the development and progression of cancer, including cervical cancer (CC). In this study, we found a CC-related lncRNA, KCNMB2-AS1, which was significantly overexpressed in CC and linked to poor outcomes. Depletion of KCNMB2-AS1 remarkably inhibited CC cell proliferation and induced apoptosis. In vivo xenograft models revealed that knockdown of KCNMB2-AS1 evidently delayed tumor growth. Mechanistically, KCNMB2-AS1 was predominantly located in the cytoplasm and served as a competing endogenous RNA to abundantly sponge miR-130b-5p and miR-4294, resulting in the upregulation of IGF2BP3, a well-documented oncogene in CC. Moreover, IGF2BP3 was able to bind KCNMB2-AS1 by three N6-methyladenosine (m6A) modification sites on KCNMB2-AS1, in which IGF2BP3 acted as an m6A "reader" and stabilized KCNMB2-AS1. Thus, KCNMB2-AS1 and IGF2BP3 formed a positive regulatory circuit that enlarged the tumorigenic effect of KCNMB2-AS1 in CC. Together, our data clearly suggest that KCNMB2-AS1 is a novel oncogenic m6A-modified lncRNA in CC, targeting KCNMB2-AS1 and its related molecules implicate the therapeutic possibility for CC patients.
Assuntos
Proliferação de Células/fisiologia , Neoplasias do Colo do Útero/metabolismo , Adulto , Animais , Western Blotting , Proliferação de Células/genética , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
OBJECTIVE: Antenatal exposure to glucocorticoids increases cardiovascular risks related to vascular dysfunctions in offspring, although underlying mechanisms are still unknown. As an important vascular mediator, high-conductance Ca2+-activated K+ channels (BK) plays an essential role in determining vascular tone. Long-term effects of antenatal glucocorticoids on BK in offspring are largely unknown. This study examined the effects and mechanisms of antenatal exposure to clinically relevant doses of glucocorticoids on vascular BK in offspring. Approach and Results: Pregnant Sprague-Dawley rats received synthetic glucocorticoids dexamethasone or vehicle during the last week of pregnancy. Vascular functions, cellular electrophysiology, target gene expression, and promoter methylation were examined in mesenteric arteries of male offspring (gestational day 21 [fetus] and postnatal day 120 [adult offspring]). Antenatal dexamethasone exposure impaired BK activators-mediated relaxation and reduced whole-cell BK currents in mesenteric arteries. Antenatal dexamethasone exposure did not alter Ca2+/voltage-sensitivity of BK but downregulated the expressions of BK α and ß1 subunits in both fetal and adult mesenteric arteries. In addition, increased promoter methylations within BKα and BKß1 were compatible with reduced expressions of the 2 genes. CONCLUSIONS: Our findings showed a profound and long-term impact of antenatal dexamethasone exposure on vascular BK via an altered epigenetic pattern from fetal stage to adulthood, advancing understanding of prolonged adverse effects and mechanisms of antenatal glucocorticoids exposure on vascular health in offspring.
Assuntos
Metilação de DNA , Dexametasona/toxicidade , Epigênese Genética , Glucocorticoides/toxicidade , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Artérias Mesentéricas/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Regiões Promotoras Genéticas , Potenciais de Ação , Animais , Dexametasona/administração & dosagem , Feminino , Glucocorticoides/administração & dosagem , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Exposição Materna , Artérias Mesentéricas/fisiopatologia , Gravidez , Ratos Sprague-Dawley , VasodilataçãoRESUMO
Large conductance of Ca2+-activated K+ channel (KCa1.1) plays an inhibitory role in neuroexcitation. However, the expression of KCNMB4/ß4-subunit in the nodose ganglia (NG) and nucleus tractus solitarius (NTS), and its effect and regulation on baroreflex afferent function at post-transcriptional level of female rats remains unknown. Here, we demonstrated that the expression of ß4-subunit encoded by KCNMB4 was significantly lower in females vs. males and ovariectomized (OVX) rats in the NG. Although all baroreceptor neurons (BRNs) expressed ß4-subunit, altered discharge characteristics were only observed in Ah-type neurons after ovariectomy. Notably, the decreased excitability of Ah-types was restored by paxilline and further enhanced by iberiotoxin. The consistent changes were observed in excitatory post-synaptic currents. The level of miR-504 was higher in females, which was predicted to bind to the 3'UTR of KCNMB4. In consistent, an inverse expression pattern between miR-504 and KCNMB4 was observed in baroreflex afferents. The paxilline-sensitive ß4-subunits is less in Ah-types and up-regulated by ovariectomy. These data indicated that KCa1.1 ß4-subunit is the key regulator in neuroexcitation of Ah-types and sexual-dimorphism in baroreflex afferent function through estrogen-dependent inhibition of KCNMB4 expression via miR-504.
Assuntos
Barorreflexo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta , MicroRNAs , Proteínas do Tecido Nervoso , Vias Aferentes , Animais , Estrogênios , Feminino , Masculino , Pressorreceptores , RatosRESUMO
OBJECTIVE: The present prospective follow-up study aimed to evaluate the effects of KCNMB2 gene polymorphisms on ritodrine efficacy and adverse drug events (ADEs) in patients with preterm labor. METHODS: A total of 163 preterm labor patients were included in this single-center study. Nine single nucleotide polymorphisms (SNPs) in the KCNMB2 gene (rs10936979, rs7624046, rs7429015, rs7625907, rs6443559, rs9839376, rs9637454, rs11918114, and rs1382045) were assessed. The primary endpoint was time to delivery, and the secondary endpoint was ritodrine-induced ADEs. RESULTS: Patients with variant homozygotes of two SNPs (rs7624046 and rs9839376), which were in linkage disequilibrium, showed 2.06 [95% confidence interval (CI), 1.14-3.73] and 2.68 (95% CI, 1.16-6.20) times the hazard of time to delivery compared to wild-type allele carriers, respectively. Among demographic characteristics, gestational age at start of drug therapy and modified Bishop score were significant factors for time to delivery. Regarding safety outcomes, patients with variant homozygotes of rs7625907 had fewer ADEs compared to those with other genotypes (odds ratio, 0.32; 95% CI, 0.13-0.83). CONCLUSION: This pharmacogenomic study suggests that ritodrine efficacy and ADEs are associated with KCNMB2 gene polymorphisms in patients with preterm labor.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Trabalho de Parto Prematuro/tratamento farmacológico , Polimorfismo de Nucleotídeo Único , Ritodrina/administração & dosagem , Tocolíticos/administração & dosagem , Adulto , Feminino , Idade Gestacional , Humanos , Desequilíbrio de Ligação , Modelos Logísticos , Idade Materna , Trabalho de Parto Prematuro/genética , Gravidez , Segundo Trimestre da Gravidez/genética , Terceiro Trimestre da Gravidez/genética , Estudos Prospectivos , Ritodrina/efeitos adversos , Tocolíticos/efeitos adversosRESUMO
The large conductance Ca2+-activated K+ (BK) channels, composed of the pore-forming α subunits (BK-α, encoded by KCNMA1 gene) and the regulatory ß1 subunits (BK-ß1, encoded by KCNMB1 gene), play a unique role in the regulation of coronary vascular tone and myocardial perfusion by linking intracellular Ca2+ homeostasis with excitation-contraction coupling in coronary arterial smooth muscle cells (SMCs). The nuclear factor erythroid 2-related factor 2 (Nrf2) belongs to a member of basic leucine zipper transcription factor family that regulates the expression of antioxidant and detoxification enzymes by binding to the antioxidant response elements (AREs) of these target genes. We have previously reported that vascular BK-ß1 protein expression was tightly regulated by Nrf2. However, the molecular mechanism underlying the regulation of BK channel expression by Nrf2, particularly at transcription level, is unknown. In this study, we hypothesized that KCNMA1 and KCNMB1 are the target genes of Nrf2 transcriptional regulation. We found that BK channel protein expression and current density were diminished in freshly isolated coronary arterial SMCs of Nrf2 knockout (KO) mice. However, BK-α mRNA expression was reduced, but not that of BK-ß1 mRNA expression, in the arteries of Nrf2 KO mice. Promoter-Nrf2 luciferase reporter assay confirmed that Nrf2 binds to the ARE of KCNMA1 promoter, but not that of KCNMB1. Adenoviral expression and pharmacological activation of Nrf2 increased BK-α and BK-ß1 protein levels and enhanced BK channel activity in coronary arterial SMCs. Hence, our results indicate that Nrf2 is a key determinant of BK channel expression and function in vascular SMCs. Nrf2 facilitates BK-α expression through a direct increase in gene transcription, whereas that on BK-ß1 is through a different mechanism.