Channel Messenger RNA Research Articles

Chlorpyrifos exposure induces calcium-dependent necrosis in carp (Cyprinus carpio) lymphocytes via the inhibition of T cell receptor gamma (TCR γ)

The insecticide chlorpyrifos plays an important role in agricultural production and is widely used because of its excellent insecticidal ability. However, the mechanism by which chlorpyrifos causes lymphocyte death remains unclear. In this study, transcriptomic techniques were used to analyze the head kidney tissues of carp (Cyprinus carpio) treated with chlorpyrifos. Subsequently, we screened out differentially expressed genes (DEGs) and performed the corresponding processing in the head kidney lymphocyte. Then, the intracellular calcium content and necrosis were detected by fluorescence staining, real-time fluorescence quantitative PCR, and flow cytometry. Our results showed that the expression of T cell receptor gamma (TCR γ) was significantly decreased, and TCR γ was inhibited after chlorpyrifos treatment. Also, TCR γ significantly increased the abundance of calcium channel messenger RNA (mRNA). To verify this result, we established the TCR γ overexpression group and found that the reverse results were observed in TCR γ of in the overexpression group. The results of cytoplasmic calcium concentration detection, calcium staining, and flow cytometry confirmed the conclusion of increased calcium in the cytoplasm. The function of TCR γ significantly enhanced the mRNA expression levels of necrosis-related genes, and this conclusion was evidenced by the result of necrotic flow detection. Our results showed that chlorpyrifos could inhibit TCR γ in carp lymphocytes and induce calcium-dependent necrosis.

Cardiac Resynchronization and Circulating Markers of Sarcoplasmic Reticulum Calcium Handling and SuddenDeath Risk.

Cardiac resynchronization therapy (CRT) can improve heart function and decrease arrhythmic events. We tested whether CRT altered circulating markers of calcium handling and sudden death risk. Circulating cardiac sodium channel messenger RNA (mRNA) splicing variants indicate arrhythmic risk, and a reduction in sarco/endoplasmic reticulum calcium adenosine triphosphatase 2a (SERCA2a) is thought to diminish contractility in heart failure. CRT was associated with a decreased proportion of circulating, nonfunctional sodium channels and improved SERCA2a mRNA expression. Patients without CRT did not have improvement in the biomarkers. These changes might explain the lower arrhythmic risk and improvedcontractility associated with CRT.

Identification of MicroRNA-Potassium Channel Messenger RNA Interactions in the Brain of Rats With Post-traumatic Epilepsy.

Background: Dysregulated expression of microRNAs and potassium channels have been reported for their contributions to seizure onset. However, the microRNA–potassium channel gene interactions in traumatic brain injury-induced post-traumatic epilepsy (PTE) remain unknown.Methods: PTE was induced in male rats by intracranial injection with ferrous chloride (0.1 mol/L, 1 μl/min) at the right frontal cortex. Electroencephalography was recorded at 60 min, as well as day 1, 7, and 30, and the behavioral seizures were assessed before injection and at different time points after injection. Rats were killed on day 30 after injection. The right frontal cortex samples were collected and subjected to high throughput messenger RNA (mRNA) and microRNA sequencing. A network of differentially expressed potassium channel mRNAs and microRNAs was constructed using OryCun2.0 and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. The differential mRNA and microRNA expressions were verified using quantitative real-time-PCR. The microRNA–mRNA was subject to the Pearson correlation analysis.Results: A PTE rat model was successfully established, as evidenced by behavioral seizures and epileptiform discharges on electroencephalography in PTE rats compared with sham rats. Among the 91 mRNAs and 40 microRNAs that were significantly differentially expressed in the PTE rat brain, 4 mRNAs and 10 microRNAs were associated with potassium channels. Except for potassium calcium-activated channel subfamily N member 2, the other three potassium channel mRNAs were negatively correlated with seven microRNAs. These microRNA–mRNA pairs were enriched in annotations and pathways related to neuronal ion channels and neuroinflammation. Quantitative real-time-PCR and correlation analysis verified negative correlations in miR-449a-5p-KCNH2, miR-98-5p-KCNH2, miR-98-5p-KCNK15, miR-19b-3p-KCNK15, and miR-301a-3p-KCNK15 pairs.Conclusion: We identified microRNA–potassium channel mRNA interactions associated with PTE, providing potential diagnostic markers and therapeutic targets for PTE.

Ion channel messenger RNA processing defects and arrhythmia

Messenger RNA (mRNA) processing is an essential step for the expression of most eukaryote genes. Ion channels are critical for proper electrical activity in the heart, and perturbations of these channels are known to cause arrhythmia. Recently, mRNA processing defects have been shown to contribute to altered ion channel activity and arrhythmogenesis. Abnormal pre-mRNA splicing of cardiac ion channels, including the cardiac sodium channel, potassium channels, and calcium channels, because of mutations of the cis-elements within the RNA or abnormal expression of splicing factors, has been documented to contribute to arrhythmic risk. In addition to pre-mRNA splicing, other mRNA processing events, such as 3'-end formation and mRNA turnover, are also disrupted in cardiac diseases, such as congenital heart disease caused by mutation at the 3'-untranslated region of GATA4. mRNA stability is also dysregulated by altered expression of microRNAs in atrial fibrillation. In this review, we discuss our current understanding of how mRNA processing defects contribute to the risk of arrhythmias and how monitoring the products of abnormal processing may lead to diagnostic tests for arrhythmic risk.

Sepsis varies arterial two-pore-domain potassium channel messenger RNA in mice

BackgroundHemodynamic changes are mainly responsible for organ failure and subsequently for the poor outcome of sepsis. Occurring macro- and micro-circulatory dysfunctions are not homogeneously distributed in the vessel beds. Especially mesenteric arterioles are subject to hypoperfusion during sepsis, and in consequence, a dysfunction of the downstream organs develops. Furthermore, impaired perfusion of the splanchnic area may cause intestinal barrier breakdown supporting the translocation of bacteria or toxins into the circulation aggravating a systemic infection and organ failure. The two-pore potassium channels (K2P channels) are responsible for setting the resting membrane potential of smooth muscle cells. Because of their sensitivity by various metabolic or humoral mediators, which are also varying during inflammatory processes, they can determine vascular resistance during sepsis. Dopamine receptors type 1 (D1R) and 2 (D2R) are assumed to be involved in the regulation of arterial tone under hypoxic conditions and are investigated too. Materials and methodsSepsis was induced in mice by the cecal ligation and puncture model. This study investigates the expression of K2P channels and the dopamine receptors at RNA level by real-time polymerase chain reaction analysis and two K2P channels at the protein level by Western blotting. ResultsThe RNA levels of K2P channels respond differently to sepsis. Although the weakly inward rectifying K+ channel 2 (TWIK 2) is not affected, TWIK-related acid-sensitive K+ channel 1 and 2 (TASK 1 and TASK 2) and TWIK-related K+ channel 1 (TREK 1) are partially downregulated during the course of the experiment. A downregulation of D1R and an upregulation of the D2R could be observed during the septic phase. ConclusionsThe changes shown could be important factors for the reduced mesenteric perfusion during sepsis.

HCN channelopathy and cardiac electrophysiologic dysfunction in genetic and acquired rat epilepsy models

Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post-status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression. Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively. Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities. These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.

Role of hippocampal sodium channel Nav1.6 in kindling epileptogenesis

Central nervous system plasticity is essential for normal function, but can also reinforce abnormal network behavior, leading to epilepsy and other disorders. The role of altered ion channel expression in abnormal plasticity has not been thoroughly investigated. Nav1.6 is the most abundantly expressed sodium channel in the nervous system. Because of its distribution in the cell body and axon initial segment, Nav1.6 is crucial for action potential generation. The goal of the present study was to investigate the possible role of changes in Nav1.6 expression in abnormal, activity-dependent plasticity of hippocampal circuits. We studied kindling, a form of abnormal activity-dependent facilitation. We investigated: (1) sodium channel protein expression by immunocytochemistry and sodium channel messenger RNA (mRNA) by in situ hybridization, (2) sodium current by patch clamp recordings, and (3) rate of kindling by analysis of seizure behavior. The initiation, development, and expression of kindling in wild-type mice were compared to Nav1.6 +/-med(tg) mice, which have reduced expression of Nav1.6. We found that kindling was associated with increased expression of Nav1.6 protein and mRNA, which occurred selectively in hippocampal CA3 neurons. Hippocampal CA3 neurons also showed increased persistent sodium current in kindled animals compared to sham-kindled controls. Conversely, Nav1.6 +/-med(tg) mice resisted the initiation and development of kindling. These findings suggest an important mechanism for enhanced excitability, in which Nav1.6 may participate in a self-reinforcing cycle of activity-dependent facilitation in the hippocampus. This mechanism could contribute to both normal hippocampal function and to epilepsy and other common nervous system disorders.

AUF1 is upregulated by angiotensin II to destabilize cardiac Kv4.3 channel mRNA

Expression of cardiac myocyte Kv4 channels (Kv4.3 for human, Kv4.2 and Kv4.3 for rodents) is downregulated with hypertrophy in vivo leading to a decrease in the transient outward current (Ito). This effect is recapitulated in vitro with rat neonatal cardiac myocytes treated with angiotensin II (Ang II), which acts via AT 1 receptors, NADPH oxidase and p38 MAP kinase to destabilize the 3′ untranslated region (3′UTR) of the Kv4.3 channel messenger RNA (mRNA). Here deletion analysis and mutagenesis identify an AU-rich element (ARE) in the Kv4.3 3′UTR that is required for Ang II-induced destabilization. Overexpression of AUF1 (ARE/poly-(U)-binding/degradation factor 1), an RNA destabilizing protein, mimics and occludes the Ang II effect, while RNA interference targeted against AUF1 blocks the Ang II effect on the Kv4.3 3′UTR. Ang II upregulates AUF1 by activating AT 1 receptors, NADPH oxidase and p38 MAP kinase. Finally, pull-down assays establish that Ang II increases AUF1 binding to the ARE required for destabilization, while binding of the mRNA stabilizing protein HuR is unaffected. Hence, Ang II acts via AT 1 receptors, NADPH oxidase and p38 MAP kinase to upregulate AUF1, which in turn binds to an ARE in the Kv4.3 3′UTR to destabilize the channel mRNA.

Time-dependent effect of pneumonectomy on alveolar epithelial fluid clearance in rat lungs

Objective: Because pneumonectomy initiates compensatory growth of the remaining lung, we determined the time-dependent effects of pneumonectomy on alveolar fluid clearance capacity. Methods: Alveolar fluid clearance capacity with the Evans blue-labeled albumin concentration was measured in rats 3 hours, 2 days, 7 days, 14 days, and 28 days after left pneumonectomy. The mechanisms responsible for the increase in alveolar fluid clearance were explored. Results: Alveolar fluid clearance in the remaining lung was normal through 7 days and then increased 14 and 28 days after pneumonectomy. The increase in alveolar fluid clearance at 28 days after pneumonectomy was accounted primarily by an increase in amiloride-sensitive transport. The expression of epithelial sodium channel messenger RNA was increased in the remaining lung and in type II alveolar epithelial cells isolated from rats 28 days after pneumonectomy. The number of isolated type II cells was larger in pneumonectomized rats than in control rats. Also, β-adrenergic agonist therapy increased the rate of alveolar fluid clearance at the 3-hour and 28-day time points. Conclusions: The capacity to remove alveolar fluid in the remaining lung is maintained at a normal level for up to 7 days after pneumonectomy in a rat, and then there is a marked increase in amiloride-sensitive alveolar fluid transport capacity that might depend, at least in part, on increased expression of epithelial sodium channels in type II cells and in part on the increased number of type II cells.J Thorac Cardiovasc Surg 2002;124:668-74

Myocardial hypertrophy in transgenic mice overexpressing human interleukin 1α

Background: Interleukin (IL)-1 has profound effects on nonimmune cells and organs, including the heart. The effects of IL-1 on transgenic hearts have not yet been described. Methods and Results: We generated transgenic mice overexpressing the human IL-1 gene under control of the cytomegalovirus enhancer/chicken β-actin promoter. Heart weight–body weight ratio increased 1.4- to 2.2-fold in transgenic mice compared with wild-type mice. Lung weight–body weight ratio also increased in transgenic mice, all of which died within 14 days of birth. Light microscopy revealed concentric hypertrophy with cardiomyocyte hypertrophy in all transgenic mice and pulmonary edema in some of them. Electron microscopy showed myofilament loss and an increased number of giant mitochondria, but no sarcomere disarray. Northern blotting showed that gene expression had been reprogrammed in the left ventricle of transgenic mice. Expression of fetal-type genes such as prepro–atrial natriuretic factor and β-myosin heavy chain were increased, but voltage-dependent calcium channel messenger RNA expression was decreased in the left ventricle of transgenic mice compared with that of wild-type mice. Conclusions: IL-1 may cause structural and functional alterations in cardiac myocytes.

Effect of prolonged O 2 deprivation on Na + channels: differential regulation in adult versus fetal rat brain

Neuronal Na + channels are functionally inhibited in the adult in response to acute O 2 deprivation. Since prolonged hypoxia may not only affect channel function, but also its expression, we hypothesized that long-term hypoxia alters Na + channel density. This alteration may depend on age, because we have found major differences in neuronal responses to hypoxia between the immature and adult. In the present work, we used northern blots, slot blots, saxitoxin binding and autoradiography to ask whether: (i) prolonged hypoxia alters Na + channel messenger RNA and protein levels in the brain; (ii) there is a difference between the adult and prenatal brains regarding Na + channel expression with hypoxic exposure; and (iii) regional differences in Na + channel expression occur in hypoxia-exposed brains. Our results show the following. (1) Na + channel messenger RNA and saxitoxin binding density decreased after prolonged hypoxia in adult brain homogenates; this is in sharp contrast to the changes observed in fetal brains, which tended to increase Na + channel messenger RNA and protein after hypoxia. (2) Changes in saxitoxin binding density are related to alterations in the number of saxitoxin binding sites and not to binding affinity, since there was no major change in K d values between the hypoxia and naive groups. (3) The hypoxia-induced Na + channel expression was heterogeneous, with major differences between rostral regions (e.g., the cortex) and caudal regions (e.g., the medulla and pons). We speculate that down-regulation of Na + channels during long-term hypoxia in mature brains is an adaptive cellular response, aimed at minimizing the mismatch between energy supply and demand, since maintenance of Na + gradients is a major energy-requiring process. However, the prenatal brain does not depend on this adaptive mechanism in response to hypoxic stress.

High-voltage-activated calcium channel messenger RNA expression in the 140-3 neuroblastoma–glioma cell line

Expression of calcium channel α 1 subunits in oocytes or cell lines has proven to be a powerful method in the analysis of structure–function relations, but these experimental systems are of limited value in the examination of neuron-specific functions such as transmitter release. Cell lines derived from neurons are often capable of these functions, but their intrinsic calcium channel α 1 subunits are complicating factors in experimental design. We have examined the biophysical and molecular properties of calcium channels in a little studied neuroblastoma–glioma hybrid cell line, 140-3, a close relative of the NG108-15 cell line, to test whether this cell line might serve a role as an expression system for neural mechanisms. This cell was selected as it contains an intact transmitter release mechanism yet secretes little in response to depolarization. Patch-clamp recording revealed only a prominent low-threshold, rapidly inactivating calcium current with a single-channel conductance of ∼7 pS that was identified as T type. A search for calcium channel α 1 subunit messenger RNA in the 140-3 cells with three different tests only revealed α 1C, whereas α 1A–α 1C were present in the parent NG108-15 line. We made a particular effort to search for α 1E, since this subunit has been associated with a low-voltage-activated current. Our findings suggest that, since the principal nerve terminal-associated calcium channels (α 1A, α 1B, α 1E) are absent in the 140-3 cell, this cell line may prove a particularly useful model for the analysis of the role of high-voltage-activated calcium channels in complex functions of neuronal cells.

Dexamethasone rapidly increases calcium channel subunit messenger RNA expression and high voltage-activated calcium current in clonal pituitary cells.

Glucocorticoid hormones increase voltage-gated Ca(2)+ current density in clonal pituitary cells. To test whether these steroids might stimulate expression of Ca(2)+ channel genes, messenger RNase protection assays were used to measure alpha IC and alpha ID RNAs that encode pore-forming subunits of L-type Ca2+ channels. We show here that dexamethasone rapidly increases alpha IC messenger RNA expression without affecting alpha ID messenger RNA level. This up-regulation of channel messenger RNA is also produced by natural glucocorticoids and is blocked by the glucocorticoid antagonist Ru48386. The up-regulation of the channel subunit messenger RNA expression is associated with an increase in high voltage-activated Ca(2)+ current density. Thus, glucocorticoids may produce a long-term effect on Ca(2)+ homeostasis in clonal pituitary cells by differentially regulating expression of Ca(2)+ channel subunit genes.

Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.

Differential up-regulation of voltage-dependent na + channels induced by phenytoin in brains of genetically seizure-susceptible (el) and control (ddy) mic

We investigated the effect of in vivo administration of an antiepileptic drug, phenytoin, on the saxitoxin binding capacity of receptor site 1 of the Na + channel α-subunit, and the expression activity of the channel messenger RNA in epileptic El mouse brains, as compared with parental ddY mice. Subchronic treatment with phenytoin (25 mg/kg per day) for 14 days increased the [ 3H]saxitoxin binding to brain-derived synaptic membranes of both El and control ddY mice in a time dependent manner. This increase plateaued at 21 ± 4% in El mice and 28 ± 3% in ddY control mice after administration of phenytoin for seven days. After cessation of treatment with phenytoin, [ 3H]saxitoxin binding capacity returned to the basal level within two weeks in both ddY and El brains. Scatchard plot analysis revealed that the phenytoin treatment caused a 20–30% increase in maximum binding capacity of [ 3H]saxitoxin binding without any change in equilibrium dissociation constant in the brain cortical synaptic membranes of both epileptic El and control ddY mice. A single injection of phenytoin (25 mg/kg) elevated the level of Na + channel messenger RNA within l h in ddY mouse brains. The increase in Na + channel messenger RNA reached a peak (about 80% increase) after 5 h of phenytoin administration in a concentration-dependent manner (6.25–50 mg/kg). On the other hand, in El mouse brains, Na + channel messenger RNA was not elevated until more than 5 h after phenytoin injection, and was increased by only about 33%. These results indicate that subchronic administration of phenytoin enhanced the expression of voltage-dependent Na + channel messenger RNA, which was followed by an increase in the number of Na + channels in both El and ddY mice. However, a single injection of phenytoin revealed a delayed expression of Na + channel messenger RNA in El mouse brain, compared to ddY brains, suggesting that the molecular basis for up-regulation of Na + channels in epileptic El brains is abnormal.

Dexamethasone Increases Potassium Channel Messenger RNA and Activity in Clonal Pituitary Cells

Glucocorticoid hormones are released as part of the stress response and regulate secretion by the pituitary. Since the activity of ion channels also influences secretion, we examined the effect of the glucocorticoid agonist dexamethasone on ion channel expression. K+ channel mRNA was detected in rat hypothalamus and anterior pituitary, with probes derived from the rat Kv1 gene, a member of the mammalian voltage-gated K+ channel superfamily. High levels were also detected in PRL-secreting clonal (GH3 and GH4C1) rat pituitary cells. Dexamethasone rapidly increased the steady state concentration of Kv1 mRNA in GH3 cells in a dose-dependent manner. This change in gene expression was accompanied by an increase in whole cell voltage-gated K+ current [lk(i)] with similar pharmacology to the Kv1 gene product. Our findings indicate that hormones may act directly on excitable cells to produce long term effects on electrical activity and secretion by regulating K+ channel expression.

Hormonal regulation of K+-channel messenger RNA in rat myometrium during oestrus cycle and in pregnancy

The dramatic enhancement of smooth-muscle excitability in the uterus which occurs at oestrus and at term in pregnant rats is closely related to increased blood oestrogen concentrations. How oestrogen alters the electrical properties of myometrial cells is unclear, although electrical coupling between cells has been shown to increase. Many examples are known of changes in cellular excitability involving modification of existing ion channels by second-messenger pathways. Steroid hormones, in contrast, are generally thought to influence cellular processes mainly through effects on gene expression, inducing the synthesis of new proteins. Previous work, using an oocyte translation system, has shown that a very slowly activating, voltage-dependent K+ current can be expressed from the poly(A)+ RNA of oestrogen-treated rat uteri. We report here that the messenger RNA species producing this channel is rapidly and reversibly induced in the presence of oestrogen, as shown by the appearance and disappearance of this mRNA during the oestrous cycle, its emergence at the end of pregnancy, and its presence or absence following hormonal treatments. These results suggest that oestrogen controls the expression of a voltage-dependent ion channel in uterine smooth muscle cells.