The Alternatively Spliced Form "b" of the Epithelial Sodium Channel α subunit (α ENaC): Any Prior Evidence of its Existence?

The epithelial sodium channel (ENaC) is critical in maintaining sodium balance across aldosterone-responsive epithelia. ENaC is a combined channel formed of three subunits (αßγ) with α ENaC subunit being the most critical for channel functionality. In a previous report, we have demonstrated the existence and mRNA expression levels of four alternatively spliced forms of the α ENaC subunit denoted by -a, -b, -c and -d in kidney cortex of Dahl S and R rats. Of the four alternatively spliced forms presently identified, α ENaC-b is considered the most interesting for the following reasons: Aside from being a salt-sensitive transcript, α ENaC-b mRNA expression is ∼32 fold higher than α ENaC wildtype in kidney cortex of Dahl rats. Additionally, the splice site used to generate α ENaC-b is conserved across species. Finally, α ENaC-b mRNA expression is significantly higher in salt-resistant Dahl R rats versus salt-sensitive Dahl S rats. As such, this commentary aims to highlight some of the previously published research articles that described the existence of an additional protein band on α ENaC western blots that could account for α ENaC-b in other rat species.

The Epithelial Sodium Channel alpha subunit (alpha ENaC) alternatively spliced form "b" in Dahl rats: What's next?

BACKGROUND: The amiloride-sensitive Epithelial Sodium Channel (ENaC) is critical in maintaining Na+ balance, extracellular fluid volume and long term blood pressure control. ENaC is composed of three main subunits alpha, beta, & gamma. While alpha ENaC is critical for channel functionality, beta & gamma ENaC maximize channel function. To date, there are four alternatively spliced forms of the alpha subunit of ENaC (alpha ENaC-a, -b, -c, & -d) that have been published in rats, in addition to the major alpha ENaC transcript. While alpha ENaC-a, -c & -d transcripts are low abundance transcripts compared to full-length alpha ENaC, alpha ENaC-b is a higher abundance and salt-sensitive transcript compared to full-length alpha ENaC. PRESENTATION OF THE HYPOTHESIS: alpha ENaC-b protein, which is preferentially produced in Dahl R rats, to a greater extent on high salt diet, exerts a dominant negative effect on full-length alpha ENaC subunit by physically binding to and trapping full-length alpha ENaC subunit in the endoplasmic reticulum, and finally accelerating full-length alpha ENaC proteolytic degradation in a dose-dependent manner. TESTING THE HYPOTHESIS: 1) To examine the mRNA and protein abundance of alpha ENaC-b relative to alpha ENaC full-length in kidney, lung, and taste tissues of Dahl rats. 2) To compare the expression (mRNA and protein) of alpha ENaC-b in kidneys of Dahl S and R rats on regular and high salt diet. 3) To examine the putative binding of alpha ENaC-b proteins to full-length alpha ENaC in vitro and to determine the impact of such binding on full-length alpha ENaC expression in vitro. IMPLICATIONS OF THE HYPOTHESIS: Our studies will be the first to demonstrate the over-expression of salt-sensitive alpha ENaC-b spliced form in kidney tissues of Dahl R rats at the expense of full-length alpha ENaC. The current proposal will provide highly novel insights into the putative mechanisms leading to ENaC hypoactivity in high-salt-fed Dahl R rats. Finally, findings from the present proposal will uncover a new mechanism by which alternative splicing may control the regulation of ENaC expression/function.

A proposed study on how far anger contributes to initiating essential hypertension in canadian caucasian adults with a family history of hypertension.

The goal of the present correlational study is to test the hypothesis that anger initiates essential hypertension in Canadian Caucasian adults with a family history of hypertension. The study population will include a cohort of 100 men and women aged 25 to 45 years at enrollment recruited from the University of Ottawa General and Civic Hospitals. Participants who are normotensives will be included in the study given that they have a family history of hypertension. The Spielberger State Trait Anger Expression inventory (STAXI) scales will assess the three main dimensions of anger: State, Trait and Anger Expression. Using a semiautomated blood pressure machine, blood pressure measurements will be conducted by the attending nurse. It is hypothesized that during the three year study, participants with higher STAXI scores will more likely develop hypertension. Results obtained from the present study are expected to highlight the significant contribution of anger as a modifiable behavioral risk factor in the pathogenesis of hypertension.

Regulation of the epithelial sodium channel [ENaC] in kidneys of salt-sensitive Dahl rats: insights on alternative splicing.

The epithelial sodium channel [ENaC] is critical for the maintenance of sodium balance, extracellular fluid volume and long term blood pressure control. Monogenic disorders causing ENaC hyperactivity have led to a severe form of hereditary hypertension in humans, known as Liddle's syndrome. Similarly, in animal models, ENaC hyperactivity has been well documented in kidneys of salt-sensitive [S] Dahl rats [a genetic model of salt-sensitive hypertension] versus their normotensive control [Dahl salt-resistant [R] rats]. The purpose of the present review is to highlight the differential regulation of ENaC in kidneys of Dahl S versus R rats. A systematic overview of the putative role of alternative splicing of the main alpha subunit of ENaC [alpha ENaC] in modulating ENaC expression in kidneys of Dahl rats will be discussed. Finally, a better understanding of the meaningful contribution of ENaC in the pathogenesis of salt-sensitive hypertension will be achieved upon completion of this review.

Characterization of the epithelial sodium channel alpha subunit coding and non-coding transcripts and their corresponding mRNA expression levels in Dahl R versus S rat kidney cortex on normal and high salt diet.

AIMS/HYPOTHESIS: The alpha subunit of the amiloride-sensitive epithelial sodium channel (alpha ENaC) is critical for the expression of functional channels. In humans and rats, non functional alternatively spliced forms of alpha ENaC have been proposed to act as negative regulatory components for ENaC. The purpose of this study was to examine the presence and consequently investigate the mRNA expression levels of alternatively spliced forms of alpha ENaC in kidney cortex of Dahl salt-resistant rats (R) versus Dahl salt-sensitive rats (S) on high salt and normal diets. METHODS: Using quantitative RT-PCR strategy, we examined the mRNA expression levels of previously reported alpha ENaC-a and -b alternatively spliced forms in kidney cortex of Dahl S and R rats on normal and four-week high salt diet and compared their corresponding abundance to wildtype alpha ENaC mRNA levels. We identified 2 novel non-coding C-terminus spliced forms and examined their mRNA expression in Dahl R versus S rat kidney cortex. We also tested the presence of five previously reported lung-specific alpha ENaC spliced forms in Dahl rat kidney cortex (CK479583, CK475461, CK364785, CK475819, and CB690980). RESULTS: Previously reported alpha ENaC-a and -b alternatively spliced forms are present in Dahl rat kidney cortex and are significantly higher in Dahl R versus S rats (P < 0.05). Four-week high salt diet significantly increases alpha ENaC-b (P < 0.05), but not alpha ENaC-a transcript abundance in Dahl R, but not S rats. Two non-coding alpha ENaC spliced forms -c and -d are newly identified in the present study, whose levels are comparable in Dahl R and S rats. Compared to alpha ENaC-wt, alpha ENaC-a, -c and -d are low abundance transcripts (4 +/- 2, 110 +/- 20, and 10 +/- 2 fold less respectively), in contrast to alpha ENaC-b abundance that exceeds alpha ENaC-wt by 32 +/- 3 fold. We could not identify any of the five previously reported lung-specific alpha ENaC spliced forms (CK479583, CK475461, CK364785, CK475819, and CB690980) in Dahl rat kidney cortex. CONCLUSION/INTERPRETATION: alpha ENaC alternative splicing might regulate alpha ENaC by the formation of coding RNA species (alpha ENaC-a and -b) and non-coding RNA species (alpha ENaC-c and -d). alpha ENaC-a and -b mRNA levels are significantly higher in Dahl R versus S rats. Additionally, alpha ENaC-b is a salt-sensitive transcript whose levels are significantly higher 4-weeks post high salt diet compared to normal salt diet in Dahl R rats. Among the four alpha ENaC transcripts (-a, -b, -c and -d), alpha ENaC-b is a predominant transcript that exceeds alpha ENaC-wt abundance by ~32 fold. alpha ENaC-a and -b spliced forms, particularly, alpha ENaC-b, might potentially act as dominant negative proteins for ENaC activity, thereby rescuing Dahl R rats from developing salt-sensitive hypertension on high salt diet. On the other hand, non-coding alpha ENaC-c and -d might assist alternative splicing, facilitate RNA processing, or regulate alpha ENaC as well as each other.

Role of the IRS-1 and/or -2 in the pathogenesis of insulin resistance in Dahl salt-sensitive (S) rats.

Insulin resistance is a common finding in hypertensive humans and animal models. The Dahl salt-sensitive (S) rat is an ideal model of genetically predetermined insulin resistance and salt-sensitive hypertension. Along the insulin signaling pathway, the insulin receptor substrates 1 and 2 (IRS-1 and -2) are important mediators of insulin signaling. IRS-1 and/or IRS-2 genetic variant(s) and/or enhanced serine phosphorylation correlate with insulin resistance. The present commentary was designed to highlight the significance of IRS-1 and/or -2 in the pathogenesis of insulin resistance. An emphasis will be given to the putative role of IRS-1 and/or -2 genetic variant(s) and serine phosphorylation in precipitating insulin resistance.

Important genetic checkpoints for insulin resistance in salt-sensitive (S) Dahl rats.

Despite the marked advances in research on insulin resistance (IR) in humans and animal models of insulin resistance, the mechanisms underlying high salt-induced insulin resistance remain unclear. Insulin resistance is a multifactorial disease with both genetic and environmental factors (such as high salt) involved in its pathogenesis. High salt triggers insulin resistance in genetically susceptible patients and animal models of insulin resistance. One of the mechanisms by which high salt might precipitate insulin resistance is through its ability to enhance an oxidative stress-induced inflammatory response that disrupts the insulin signaling pathway. The aim of this hypothesis is to discuss two complementary approaches to find out how high salt might interact with genetic defects along the insulin signaling and inflammatory pathways to predispose to insulin resistance in a genetically susceptible model of insulin resistance. The first approach will consist of examining variations in genes involved in the insulin signaling pathway in the Dahl S rat (an animal model of insulin resistance and salt-sensitivity) and the Dahl R rat (an animal model of insulin sensitivity and salt-resistance), and the putative cellular mechanisms responsible for the development of insulin resistance. The second approach will consist of studying the over-expressed genes along the inflammatory pathway whose respective activation might be predictive of high salt-induced insulin resistance in Dahl S rats. Variations in genes encoding the insulin receptor substrates -1 and/or -2 (IRS-1, -2) and/or genes encoding the glucose transporter (GLUTs) proteins have been found in patients with insulin resistance. To better understand the combined contribution of excessive salt and genetic defects to the etiology of the disease, it is essential to investigate the following question:Question 1: Do variations in genes encoding the IRS -1 and -2 and/or genes encoding the GLUTs proteins predict high salt-induced insulin resistance in Dahl S rats?A significant amount of evidence suggested that salt-induced oxidative stress might predict an inflammatory response that upregulates mediators of inflammation such as the nuclear factor- kappa B (NF-kappa B), the tumor necrosis factor-alpha (TNF-alpha) and the c-Jun Terminal Kinase (JNK). These inflammatory mediators disrupt the insulin signaling pathway and predispose to insulin resistance. Therefore, the following question will be thoroughly investigated:Question 2: Do variations in genes encoding the NF-kappa B, the TNF-alpha and the JNK, independently or in synergy, predict an enhanced inflammatory response and subsequent insulin resistance in Dahl S rats in excessive salt environment?Finally, to better understand the combined role of these variations on glucose metabolism, the following question will be addressed:Question 3: What are the functional consequences of gene variations on the rate of glucose delivery, the rate of glucose transport and the rate of glucose phosphorylation in Dahl S rats?The general hypothesis is that "high-salt diet in combination with defects in candidate genes along the insulin signaling and inflammatory pathways predicts susceptibility to high salt-induced insulin resistance in Dahl S rats".

Genetic and dietary salt contributors to insulin resistance in Dahl salt-sensitive (S) rats.

Insulin resistance has been extensively investigated during the past decade because of its proposed role in initiating a cluster of cardiovascular risk factors including hypertension. Insulin resistance is an inherited genetic trait that precedes hypertension in Dahl salt-sensitive (S) rats, and is not present in Dahl salt-resistant (R) rats. Owing to the co-existence of insulin resistance and salt sensitivity of blood pressure in Dahl S, but not R rats, Dahl S rats are used to elucidate the role of dietary salt as a potential link in exacerbating both phenotypes (insulin resistance and salt sensitivity). In light of available data, examining the impact of dietary salt on insulin resistance in Dahl S rats in terms of salt concentration and duration of exposure helps answer the following question: What percentage of dietary salt and for what duration of exposure would we expect an enhanced insulin resistance in Dahl S rats? This commentary gathers all available research done on insulin resistance in Dahl S rats in an attempt to unravel dietary salt contribution to insulin resistance in Dahl S rats.

Sequence analysis of coding and 3' and 5' flanking regions of the epithelial sodium channel alpha, beta, and gamma genes in Dahl S versus R rats.

BACKGROUND: To test whether epithelial sodium channel (ENaC) genes' variants contribute to salt sensitive hypertension in Dahl rats, we screened ENaC alpha, beta, and gamma genes entire coding regions, intron-exon junctions, and the 3' and 5' flanking regions in Dahl S, R and Wistar rats using both Denaturing High Performance Liquid Chromatography (DHPLC) and sequencing. RESULTS: Our analysis revealed no sequence variability in the three genes encoding ENaC in Dahl S versus R rats. One homozygous sequence variation predicted to result in a D75E substitution was identified in Dahl and Wistar rat ENaC alpha compared to Brown Norway. Six and two previously reported polymorphic sites in Brown Norway sequences were lost in Dahl and Wistar rats, respectively. In the 5' flanking regions, we found a deletion of 5GCTs in Dahl and Wistar rat ENaC alpha gene, five new polymorphic sites in ENaC beta and gamma genes, one homozygous sequence variation in Dahl and Wistar rat ENaC gamma gene, as well as one Dahl rat specific homozygous insertion of -1118CCCCCA in ENaC gamma gene. This insertion created additional binding sites for Sp1 and Oct-1. Five and three Brown Norway polymorphic sites were lost in Dahl and Wistar rats, respectively. No sequence variability in ENaC 3' flanking regions was identified in Dahl compared to Brown Norway rats. CONCLUSION: The first comprehensive sequence analysis of ENaC genes did not reveal any differences between Dahl S and R rats that were isogenic in the regions screened. Mutations in ENaC genes intronic sequence or in ENaC-regulatory genes might possibly account for increased ENaC activity in Dahl S versus R rats.

Fast, cheap and out of control: a zero curation model for ontology development.

During two days at a conference focused on circulatory and respiratory health, 68 volunteers untrained in knowledge engineering participated in an experimental knowledge capture exercise. These volunteers created a shared vocabulary of 661 terms, linking these terms to each other and to a pre-existing upper ontology by adding 245 hyponym relationships and 340 synonym relationships. While ontology-building has proved to be an expensive and labor-intensive process using most existing methodologies, the rudimentary ontology constructed in this study was composed in only two days at a cost of only 3 t-shirts, 4 coffee mugs, and one chocolate moose. The protocol used to create and evaluate this ontology involved a targeted, web-based interface. The design and implementation of this protocol is discussed along with quantitative and qualitative assessments of the constructed ontology.

Apoptosis effects of Xrel3 c-Rel/Nuclear Factor-kappa B homolog in human cervical cancer cells.

Cervical cancer is considered a common yet preventable cause of death in women. In this report, we studied the role of the NF-kappaB gene family in HeLa human cervical cancer cells, using the Xrel3 c-Rel homologue of Xenopus laevis. The expression of Xrel3/c-Rel slowed cell growth 6-fold, consistent with an upregulated expression of the cell cycle inhibitor p21. The activated PARP apoptosis effector was significantly increased (P<0.01). Based on cell viability assays Xrel3 provided an anti-apoptotic effect in 1 microM cisplatin, and this was associated with significantly lower levels of the apoptotic proteins Bax and MDM-2 (P<0.05). Furthermore, there was a 3-fold drop in the level of the tumor suppressor protein p53. In 5 microM cisplatin, expression of HeLa Xrel3 enhanced apoptosis by significantly increasing the expression of the apoptotic proteins Bax and MDM-2 (P<0.05). However, the tumor suppressor protein p53 showed a significant decrease (P<0.05) relative to the control. Thus, c-Rel/NF-kappaB may potentially be of clinical significance, especially in tumors exhibiting resistance to high-level chemotherapy.

Rel/Nuclear factor-kappa B apoptosis pathways in human cervical cancer cells.

Cervical cancer is considered a common yet preventable cause of death in women. It has been estimated that about 420 women out of the 1400 women diagnosed with cervical cancer will die during 5 years from diagnosis. This review addresses the pathogenesis of cervical cancer in humans with a special emphasis on the human papilloma virus as a predominant cause of cervical cancer in humans. The current understanding of apoptosis and regulators of apoptosis as well as their implication in carcinogenesis will follow. A special focus will be given to the role of Rel/NF-kappaB family of genes in the growth and chemotherapeutic treatment of the malignant HeLa cervical cells emphasizing on Xrel3, a cRel homologue.

Dual apoptotic effect of Xrel3 c-Rel/NF-kappaB homolog in human cervical cancer cells.

Cervical cancer is one of the most common cancers affecting a woman's reproductive organs. Despite its frequency and recurrence, the death rate has been declining over the past 40 years, due to early detection and treatment. In a previous report [Shehata Marlene, Shehata Marian, Shehata Fady, Pater Alan. Apoptosis effects of Xrel3 c-Rel/Nuclear factor-kappa B homolog in human cervical cancer cells. Cell Biology International, in press], we studied the role of the NF-kappaB gene family in HeLa human cervical cancer cells, using the Xrel3 c-Rel homologue of Xenopus laevis. These results showed that the expression of Xrel3/c-Rel slowed cell growth, consistent with an upregulated expression of the cell cycle inhibitor p21 and the activated poly(ADP-ribose) polymerase (PARP) apoptosis effector. However, in this report, we examined more apoptotic and anti-apoptotic factors acting upstream and downstream in apoptosis pathways after cisplatin treatment of HeLa cervical cancer cells. After 1 microM cisplatin treatment, Xrel3 had an anti-apoptotic effect, based on significantly lower levels of apoptotic proteins, including caspase-8, caspase-3 and p21. Anti-apoptotic BAG-1 isoforms were upregulated. After 5 microM cisplatin treatment, expression of HeLa Xrel3 had an apoptotic effect, based on significantly increased expression of the cell cycle inhibitor p21 and apoptotic proteins, including cleaved PARP, caspase-8, and caspase-3. However, anti-apoptotic Bcl-2 and Bcl-X(L) were elevated and the cell cycle regulator cyclin D1 was slightly upregulated with both 1 and 5 microM cisplatin treatment. The HPV E6 oncoprotein showed no significant changes. These results support previous conclusions on the potential anti-apoptotic effects of c-Rel/NF-kappaB in mild stress environments, as opposed to the apoptotic effects associated with high stress conditions [Lake BB, Ford R, Kao KR. Xrel3 is required for head development in Xenopus laevis. Development 2001; 128(2), 263-73.]. Thus, c-Rel/NF-kappaB may potentially be of clinical significance in chemotherapy.