A6 Cells Research Articles

Biochemical composition of the functional amiloride‐sensitive, hetero‐multimeric, 4ps ENaC

Epithelial sodium channels (ENaC) consist of α, β, and γ subunits. As ENaC subunit proteins are synthesized in the rough endoplasmic reticulum, they are core glycosylated and co‐assembled into a hero‐multimeric channel. As they traffic through the Golgi to the apical membrane, some subunits are further glycosylated in the Golgi because the sugars become resistant to endo‐H glycosidase digestion, a hallmark of Golgi glycosylation. Since the protease, furin is active in the trans‐Golgi and at the plasma membrane, but prostasins are active only at the plasma membrane, the ENaC α and γ subunits must be cleaved either in the trans‐Golgi or at the apical membrane or both. Apical membranes of renal A6 cells contain subunits from each maturation step, endo‐H‐resistant, endo‐H‐sensitive, cleaved, and un‐cleaved as determined by isolation of biotinylated apical subunits. Because A6 cells contain only one type of functional channel, functional channel must be formed only from a subset of these modified subunits corresponding to the different bands. Therefore, we analyzed which modified subunits make‐up functional channels using co‐immunoprecipitation and other techniques. This research is supported by DK‐037963.

Molecular analysis of translational regulation of thyroid hormone receptor alpha mRNA

Amphibian metamorphosis proceeds under the control of thyroid hormone (TH) and thyroid hormone receptor (TR). TRs are encoded by two genes, TRα and TRβ. TRβ mRNA and protein rise along with the increase in endogenous TH. On the other hand TRα mRNA increases during metamorphosis, whereas TRα protein remains constant, suggesting that post‐transcriptional events control the levels of TRα protein. But the regulation of TRα protein expression remains largely unknown. We focused on the region of the TRα 5′UTR. At first, we determined nucleotide sequences of the TRα 5′UTR in amphibians by 5′‐RACE. There are highly conserved reigions in the TRα 5′UTR among the amphibians. We found that the TRα 5′UTR significantly repressed TRα mRNA translation as a cis‐regulatory element in Xenopus laevis A6 (XLA6) cells. Moreover, the TRα 5′UTR suppressed of a downstream open reading frame translation in both reticulocyte lysate and wheat germ extract. Successive deletions of the TRα 5′UTR revealed five elements to repress downstream translation. We analyzed two of five elements by extensive point mutations. One of elements is the GC‐rich region and the other is an upstream open reading frame (uORF). Most single base mutations in the GC‐rich region severely eliminated the repression of translation, while base substitutions didn't affect translational regulation by uORF.

The excitement of multiple noradrenergic cell groups in the rat brain related to hyperbaric oxygen seizure.

The mechanism of oxygen toxicity for central nervous system and hyperbaric oxygen (HBO) seizure has not been clarified. Noradrenergic cells in the brain may contribute to HBO seizure. In this study, we defined the activation of noradrenergic cells during HBO exposure by c-fos immunohistochemistry. Electroencephalogram electrodes were pre-implanted in all animals under general anesthesia. In HBO seizure animals, HBO was induced with 5 atm of 100% oxygen until manifestation of general tonic convulsion. HBO non-seizure animals were exposed to 25 min of HBO. Control animals were put in the chamber for 120 min without pressurization. All animals were processed for c-fos immunohistochemical staining. All animals in the HBO seizure group showed electrical discharge on EEG. In the immunohistochemistry, c-fos was increased in the A1, A2 and A6 cells of the HBO seizure group, and in the A2 and A6 cells of the HBO non-seizure group, yet was extremely low in all three cell types in the control group. These results suggest the participation of noradrenaline in HBO seizure, which can be explained by the early excitement of A1 cells due to their higher sensitivity to high blood pressure, hyperoxia, or by the post-seizure activation of all noradrenergic cells.

Curcumin-induced inhibition of proteasomal activity, enhanced HSP accumulation and the acquisition of thermotolerance in Xenopus laevis A6 cells

In the present study, curcumin, a phenolic compound with anti-inflammatory, anti-tumor and anti-amyloid properties, inhibited proteasomal activity and induced the accumulation of HSPs in the frog model system, Xenopus laevis. Treatment of A6 kidney epithelial cells with curcumin enhanced ubiquitinated protein levels and inhibited chymotrypsin-like activity. Furthermore, exposure of cells to 10–50μM curcumin for 24h induced HSP30 and HSP70 accumulation. This phenomenon was controlled at the transcriptional level since pre-treatment of cells with KNK437, a heat shock factor 1 (HSF1) inhibitor, repressed HSP accumulation. Additionally, elevation of the incubation temperature from 22 to 30°C greatly enhanced the curcumin-induced accumulation of HSP30 and HSP70. Immunocytochemical analysis revealed that curcumin-induced HSP30 was detectable primarily in the cytoplasm in a punctate pattern with minimal detrimental effects on the actin cytoskeleton. Finally, prior exposure of cells to curcumin conferred a state of thermotolerance since it protected them against a subsequent cytotoxic thermal challenge. These findings are of importance given the interest in identifying agents that can upregulate HSP levels with minimal effects on cell structure or function as a therapeutic treatment of protein folding diseases.

Simultaneous Noncontact Topography and Electrochemical Imaging by SECM/SICM Featuring Ion Current Feedback Regulation

We described a hybrid system of scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM) with ion current feedback nanopositioning control for simultaneous imaging of noncontact topography and spatial distribution of electrochemical species. A nanopipette/nanoring electrode probe provided submicrometer resolution of the electrochemical measurement on surfaces with complex topology. The SECM/SICM probe had an aperture radius of 220 nm. The inner and outer radii of the SECM Au nanoring electrode were 330 and 550 nm, respectively. Characterization of the probe was performed with scanning electron microscopy (SEM), cyclic voltammetry (CV), and approach curve measurements. SECM/SICM was applied to simultaneous imaging of topography and electrochemical responses of enzymes (horse radish peroxidase (HRP) and glucose oxidase (GOD)) and single live cells (A6 cells, superior cervical ganglion (SCG) cells, and cardiac myocytes). The measurements revealed the distribution of activity of the enzyme spots on uneven surfaces with submicrometer resolution. SECM/SICM acquired high resolution topographic images of cells together with the map of electrochemical signals. This combined technique was also applied to the evaluation of the permeation property of electroactive species through cellular membranes.

Manipulating Heat Shock Factor-1 in Xenopus Tadpoles: Neuronal Tissues Are Refractory to Exogenous Expression

BackgroundThe aging related decline of heat shock factor-1 (HSF1) signaling may be causally related to protein aggregation diseases. To model such disease, we tried to cripple HSF1 signaling in the Xenopus tadpole.ResultsOver-expression of heat shock factor binding protein-1 did not inhibit the heat shock response in Xenopus. RNAi against HSF1 mRNA inhibited the heat shock response by 70% in Xenopus A6 cells, but failed in transgenic tadpoles. Expression of XHSF380, a dominant-negative HSF1 mutant, was embryonic lethal, which could be circumvented by delaying expression via a tetracycline inducible promoter. HSF1 signaling is thus essential for embryonic Xenopus development. Surprisingly, transgenic expression of the XHSF380 or of full length HSF1, whether driven by a ubiquitous or a neural specific promoter, was not detectable in the larval brain.ConclusionsOur finding that the majority of neurons, which have little endogenous HSF1, refused to accept transgene-driven expression of HSF1 or its mutant suggests that HSF1 levels are strictly controlled in neuronal tissue.

WNK4 inhibits ENaC activity and reduces γ ENaC subunit expression, but has no effect on β ENaC expression

Recently, a serine‐threonine protein kinase WNK4 expressed in the distal nephron has been shown to inhibit ENaC activity; however, the mechanism of this inhibition is unclear. We examined the effect of expressing WNK4 on endogenous ENaC in A6 cells. Single channel recording shows that WNK4 inhibits ENaC activity by reducing apical channel number rather than by changing open probability. Western blots of total as well as apical biotin‐labeled ENaC β and γ subunits show that the γ subunit but not the β subunit is reduced in WNK4 expressing cells. Amiloride‐sensitive trans‐epithelial Na+ current (Isc) of WNK4 expressing cells treated with forskolin increases by the same percentage amount from baseline as forskolin‐treated, sham transfected cells; however, the maximum current amplitude of WNK4 expressing cells is about half of the control likely due to the lower basal ENaC activity. The time course of Isc after forskolin application and removal are the same in control and WNK4 expressing cells, which implies that WNK4 has no effect on PKA stimulated ENaC recycling. Interestingly, even though forskolin increased Isc of control and WNK4 expressing cells, apically expressed β ENaC does not change in either group of cells. Our data imply that WNK4 may regulate the three ENaC subunits in non‐coordinated way, which is consistent with other's reports that the apical residency of ENaC is non‐coordinately regulated.

UCH‐L3 upregulates the epithelial sodium channel in A6 distal nephron cells

Recent studies have shown that the epithelial sodium channel (ENaC) is down‐regulated via its ubiquitination induced by Nedd4‐2 and is upregulated via its deubiquitination induced by Ubiquitin Carboxyl‐Terminal Hydrolase (UCH) L3. In the present study, we used patch‐clamp techniques combined with both confocal microscopy and Western blot methods to further determine how ENaC in A6 distal nephron cells is regulated by UCH‐L3. Patch‐clamp data showed that decreasing the activity of UCH‐L3 with a UCH‐L3 inhibitor significantly reduced the number of active ENaC in the patch. The data from Western blot experiments showed that treatment of A6 cells with the UCH‐L3 inhibitor remarkably reduced the protein levels of α‐, β‐, and γ‐ENaC subunits. Consistent with these data, confocal microscopy experiments showed that the UCH‐L3 inhibitor also reduced the density of α‐ and γ‐ENaC at the apical membrane. These results suggest that UCH‐L3 upregulates ENaC by maintaining its density at the apical membrane.

Role of P97 protein in ENaC recycling

Functional Epithelial Sodium Channels, ENaC, consist of α, β, and γ subunits. A major way to regulate ENaC activity is by controlling the number of ENaC at the apical membrane by either removing ENaC from or inserting ENaC into the cell's apical membrane. Apical ENaC number can be altered by changing the rate of recycling or degradation of internalized ENaC. ENaC, a putative trimer, appears to be internalized as an ubiquitinated multimer. Some internalized ENaC are de‐ubiquitinated by endosomal enzymes and returned to the apical membrane as functional ENaC, but some are finally degraded by the proteasome, an organelle that recognizes and degrades monomeric proteins. Multimeric ENaC must therefore be disassembled before degradation. Disassembly of a multimeric protein requires significant energy that can be provided by a novel protein ATPase, P97. P97 also chaperones individual subunits of ubiquitinated proteins to the proteasome. We elucidated the role of P97 in ENaC recycling by confirming physical interaction between P97 and ENaC subunits by co‐immunoprecipitation. The impact of P97 on ENaC was determined in A6 cells over‐expressing a putative dominant negative P97. ENaC subunit amounts in whole‐cell lysates increased consistent with subunit degradation in these cells.

Celastrol can inhibit proteasome activity and upregulate the expression of heat shock protein genes, hsp30 and hsp70, in Xenopus laevis A6 cells

In eukaryotes, the ubiquitin–proteasome system (UPS) is responsible for the degradation of most proteins. Proteasome inhibition, which has been associated with various diseases, can cause alterations in various intracellular processes including the expression of heat shock protein ( hsp) genes. In this study, we show that celastrol, a quinone methide triterpene and anti-inflammatory agent, inhibited proteasome activity and enhanced HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Treatment of cells with celastrol induced the accumulation of ubiquitinated protein and inhibited chymotrypsin-like activity. This was accompanied by a dose- and time-dependent accumulation of HSP30 and HSP70. Celastrol-induced HSP accumulation was mediated by HSF1–DNA binding activity since this response was inhibited by the HSF1 activation inhibitor, KNK437. Simultaneous exposure of cells with celastrol plus either mild heat shock or the proteasome inhibitor, MG132, produced an enhanced accumulation of HSP30 that was greater than the sum of the individual stressors alone. Immunocytochemical analysis revealed that celastrol-induced HSP30 accumulation occurred in the cytoplasm in a granular pattern supplemented with larger circular HSP30 staining structures. HSP30 was also noted in the nucleus with less staining in the nucleolus. In some cells, celastrol induced the collapse of the actin cytoskeleton and conversion to a rounder morphology. In conclusion, this study has shown that celastrol inhibited proteasome activity and induced HSF1-mediated expression of hsp genes in amphibian cells.

5‐Stabilized Phosphatidylinositol 3,4,5‐Trisphosphate Analogues Bind Grp1 PH, Inhibit Phosphoinositide Phosphatases, and Block Neutrophil Migration

Metabolically stabilized analogues of PtdIns(3,4,5)P3 have shown long-lived agonist activity for cellular events and selective inhibition of lipid phosphatase activity. We describe an efficient asymmetric synthesis of two 5-phosphatase-resistant analogues of PtdIns(3,4,5)P3, the 5-methylene phosphonate (MP) and 5-phosphorothioate (PT). Furthermore, we illustrate the biochemical and biological activities of five stabilized PtdIns(3,4,5)P3 analogues in four contexts. First, the relative binding affinities of the 3-MP, 3-PT, 5-MP, 5-PT, and 3,4,5-PT3 analogues to the Grp1 PH domain are shown, as determined by NMR spectroscopy. Second, the enzymology of the five analogues is explored, showing the relative efficiency of inhibition of SHIP1, SHIP2, and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the greatly reduced ability of these phosphatases to process these analogues as substrates as compared to PtdIns(3,4,5)P3. Third, exogenously delivered analogues severely impair complement factor C5a-mediated polarization and migration of murine neutrophils. Finally, the new analogues show long-lived agonist activity in mimicking insulin action in sodium transport in A6 cells.

Analysis of Blocker-labeled Channels Reveals the Dependence of Recycling Rates of ENaC on the Total Amount of Recycled Channels

Trafficking is one of the primary mechanisms of epithelial Na⁺ channel (ENaC) regulation. Although it is known that ENaCs are recycled between the apical membrane and the intracellular channel pool, it has been difficult to investigate the recycling of ENaCs; especially endogenously expressed ENaCs. The aim of the present study is to investigate if the recycling rates of ENaCs depend on the total amount of recycled ENaCs. To accomplish this point, we established a novel method to estimate the total amount of recycled ENaCs and the ENaC recycling rates by using a specific blocker (benzamil) of ENaC with a high-affinity for functional label of the channels in recycling. Applying this method, we studied if a decrease in the total amount of ENaCs caused by brefeldin A (5 µg/mL, 1 h) affects respectively the rates of insertion and endocytosis of ENaCs to and from the apical membrane in monolayers of renal epithelial A6 cells. Our observations indicate that: 1) both insertion and endocytosis rates of ENaC increase when the total amount of ENaCs decreases, 2) the increase in the insertion rate is larger than that in the endocytosis rate, and 3) this larger increase in the insertion rate than the endocytosis rate caused by the decrease in the total amount of ENaCs plays an important role in preventing Na⁺ transport from drastically diminishing due to a decrease in the total amount of ENaCs. The newly established analysis of blocker-labeled ENaCs in the present study provides a useful tool to investigate the recycling of endogenously expressed ENaCs.

Effect of calcium oxalate on renal cells as revealed by real-time measurement of extracellular oxidative burst

Calcium oxalate is one of the main constituents of kidney stones and has a proved deleterious effect on renal cells that is mediated by oxidative stress. However, the subcellular source of this oxidative stress, and whether it is extending to the extracellular space or not, is still disputed. Therefore, an electrochemical superoxide biosensor was constructed, positioned above A6 renal cells, and used to measure in real-time the extracellular oxidative burst following addition of calcium oxalate crystals. It was observed that A6 cells do secrete superoxide into their extracellular space in few minutes after encountering calcium oxalate crystals. The amount of released superoxide peaks at about 20min. Superoxide is cleared away from the extracellular space after approximately 3h. Superoxide secretion depends on the presence of superoxide-scavenging enzyme superoxide dismutase, the age of the cells, the amount of calcium oxalate crystals, and the temperature. Moreover, the effect of calcium oxalate crystals was mimicked by phorbol 12-myristate 13-acetate. The developed sensing system can be a useful tool for biologists investigating nephrolithiasis at cellular level.

Dual transcriptional regulation by runx2 of matrix Gla protein in Xenopus laevis

Matrix Gla protein (MGP) is an extracellular mineral-binding protein expressed in several tissues but it only accumulates in bone and calcified cartilage under physiological conditions. Available evidence indicates that it acts as a physiological inhibitor of mineralization. Runx2 is a transcription factor essential for bone formation in mammals, affecting osteoblast and chondrocyte differentiation by regulating key genes crucial for bone and cartilage development. Being an important cartilage-associated gene, MGP is a potential target for Runx2, and thus we have investigated the possible functional interactions between them. In A6 cells, Runx2 was found to modulate MGP transcription and deletion analysis of MGP distal and proximal promoter-luciferase constructs identified cis-regulatory regions. Interestingly, we have also identified a runx2-binding site that mediates transcriptional repression of XlMGP. Mutation of this element, located between − 54 and + 33 bp, results in 18-fold up-regulation of transcription. Furthermore, and in addition to the previously reported Xlrunx2 types I and II, we have identified three transcripts encoding novel, truncated Xlrunx2 isoforms. Although only type I and type II could transactivate XlMGP, the truncated isoforms identified in this study, which result from alternative splicing, could be involved in negative regulation of MGP expression, as described for other RUNX2 truncated isoforms acting in other target genes. In vivo microinjection of XlMGP promoter constructs and runx2 mRNA confirmed that those promoters are targets for this transcription factor. These data also indicate that MGP is under dual regulation by runx2 through the use of various isoforms and context-dependent formation of transcriptional complexes.

Proteasome inhibition induces hsp30 and hsp70 gene expression as well as the acquisition of thermotolerance in Xenopus laevis A6 cells

Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose- and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin- or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge.

Androgen receptor of the frog Rana rugosa: molecular cloning and its characterization

The androgen receptor(AR) gene is located on the Z and W sex chromosomes in the frog Rana rugosa, designated Z- and W-AR, respectively. Among various tissues of an adult frog, AR expression levels were highest in the testis and brain. In the testis, AR was expressed in germ cells. AR expression occured in developing embryos from stage 21 and was very high in the gonad of a male tadpole before the onset of sex determination. When Z- and W-AR were expressed in Xenopus A6 cells, they activated androgen-dependent transcription of a luciferase reporter gene. By contrast, estrogen receptor (ER) alpha and beta showed no sexually dimorphic expression during sex determination, but their expressions became much higher in the gonad of a female tadpole after sex determination. In addition, AR transcripts in the ZZ-tadpoles were twice as abundant as in the ZW genotype. In contrast, W-AR expression was extremely low although when W-AR was expressed in A6 cells, it activated transcription in the luciferase assay. In this regard it is worth noting that the promoter regions of Z- and W-AR are not identical. The results suggest that Z-AR plays an important role in the testis formation in a R. rugosa tadpole, whereas ERbeta is involved in ovary differentiation. Very low expression of W-AR may be due to its promoter region having mutations in key transcription factor binding sites, although these remain to be identified. Thus, it is proposed that AR could be a candidate for a male-determining gene in R. rugosa.

Hsp70 is required for optimal cell proliferation in mouse A6 mesoangioblast stem cells

Mouse Hsp70 (70 kDa heat shock protein) is preferentially induced by heat or stress stimuli. We previously found that Hsp70 is constitutively expressed in A6 mouse mesoangioblast stem cells, but its possible role in these cells and the control of its basal transcription remained unexplored. Here we report that in the absence of stress, Ku factor is able to bind the HSE (heat shock element) consensus sequence in vitro, and in vivo it is bound to the proximal hsp70 promoter. In addition, we show that constitutive hsp70 transcription depends on the co-operative interaction of different factors such as Sp1 (specificity protein 1) and GAGA-binding protein with Ku factor, which binds the HSE consensus sequence. We used mRNA interference assays to select knockdown cell clones. These cells were able to respond to heat stress by producing a large amount of Hsp70, and produced the same amount of Hsp70 as that synthesized by stressed A6 cells. However, severe Hsp70 knockdown cells had a longer duplication time, suggesting that constitutive Hsp70 expression has an effect on the rate of proliferation.

Sox3: A transcription factor for Cyp19 expression in the frog Rana rugosa

Cyp19 is expressed at a high level in the gonad of the female tadpole of the frog Rana rugosa during sex determination. To identify sequence elements important for expression of Cyp19, we isolated a genomic clone (∼ 40 kbp) carrying R. rugosa Cyp19 and analyzed the nucleotide sequence of the 5′-flanking region to search for potential transcription factor binding sites. Sox (SRY-related HMG box) protein and Sf1 binding sites were found in the ovary-specific promoter region of Cyp19. Because Sox3 is located on the sex chromosome in R. rugosa, we conducted the luciferase reporter assay in Xenopus A6 cells using the promoter region. Sox3 drove the reporter gene in the cells, but Sf1 did not. When sequential deletion of the 2.7 kbp Cyp19-promoter region was undertaken, a fragment spanning nucleotides − 191 to + 48 was sufficient to drive the transcription of the reporter gene. In site-directed mutagenesis, the binding site at − 57 in the region was critical for Sox3 responsiveness. Sox3 lacking the HMG box had no ability to promote Cyp19 transcription. In addition, a chromatin immunoprecipitation (ChIP) assay showed that DNA fragments were enriched 8-fold, as determined by real-time PCR, when chromatin was immunoprecipitated with the anti-His antibody against His-tagged Sox3. The results, taken together, suggest that Sox3 activates Cyp19 transcription by its direct binding to the binding site of the Cyp19 promoter region. Sox3 appears to be a factor that directs indifferent gonads to develop into an ovary in R. rugosa.

Benzamil prevents brain water accumulation in hyponatraemic rats

It has been recently shown that A6 cells exposed to hyponatraemic stress respond with increased sodium uptake via activation of benzamil-sensitive sodium channels. This study was performed, therefore, to explore the possible involvement of benzamil-sensitive sodium channels and cellular sodium influx in brain oedema formation in hyponatraemic rats. Four groups of male Wistar rats were studied (n = 13 in each group). Animals in group I with normonatraemia received intracerebroventricular (icv) 0.9% NaCl; animals in group II-IV were made hyponatraemic by intraperitoneal administration of isotonic glucose solution in a dose of 20% per body weight. Rats were pretreated with icv 0.9% NaCl (group II), 120 microg arginine vasopressin (AVP) (group III) or 4 microg benzamil-hydrochloride (group IV). Plasma sodium (ion-selective electrode) plasma osmolality (vapour pressure osmometer) and brain sodium and potassium content (flame photometer) as well as brain water content (desiccation method) were measured after a 2-h hydration period. Plasma sodium, osmolality and tissue sodium and potassium contents were markedly depressed in hyponatraemic rats (group II-IV, p < 0.0005 for each group) irrespective of drug pretreatment. Brain water content, however, responded to hyponatraemia with an increase from 77.55 +/- 1.00% to 78.45 +/- 0.94% (p < 0.01), and it was further augmented to 79.35 +/- 0.80% (p < 0.0005) by icv AVP pretreatment. By contrast, benzamil administration prevented the rise of brain water caused by hyponatraemia (77.61 +/- 1.04%). Early in the course of hyponatraemia, brain sodium channels may be activated, and the subsequent cellular sodium uptake may generate osmotic gradient to allow passive water flow into the cells. The simultaneous reduction of osmotic water conductivity of brain-specific aquaporin-4 by hyponatraemia, however, may limit water accumulation.

Simultaneous exposure of Xenopus A6 kidney epithelial cells to concurrent mild sodium arsenite and heat stress results in enhanced hsp30 and hsp70 gene expression and the acquisition of thermotolerance

In this study, we examined the effect of concurrent low concentrations of sodium arsenite and mild heat shock temperatures on hsp30 and hsp70 gene expression in Xenopus A6 kidney epithelial cells. RNA blot hybridization and immunoblot analysis revealed that exposure of A6 cells to 1–10 µM sodium arsenite at a mild heat shock temperature of 30 °C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 µM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26 °C with larger responses at 28 and 30 °C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 µM) and heat shock (30 °C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge (37 °C). Acquired thermotolerance was not observed with cells treated with the two mild stresses individually.