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Abstract Background MT1-MMP is a cell-surface enzyme whose regulation of pro-MMP-2 and ERK activation position it as a key facilitator of ECM remodelling and cell migration. These processes are modulated by endogenous MMP inhibitors, such as RECK, a GPI-anchored protein which has been shown to inhibit both MT1-MMP and MMP-2 activity. Our previous studies have revealed a link between MT1-MMP levels, and pro-MMP-2 and ERK activation in mammalian cells, as well as MT1-MMP and RECK co-localization in Xenopus embryos. We here investigated how modulation of RECK would impact MT1-MMP and MMP-2 levels, as well as ERK signalling in Xenopus A6 cells. Results We used a Morpholino approach to knockdown RECK, plasmid transfection to overexpress RECK, and PI-PLC treatment to shed RECK from the cell surface of Xenopus A6 cells. RECK reduction did not alter pERK or MT1-MMP levels, nor MMP-2 activity as measured by zymography; thus RECK-knockdown cells maintained the ability to remodel the ECM. RECK overexpression and PI-PLC treatment both increased ECM remodelling potential through increased MT1-MMP protein and relative MMP-2 activation levels. Conclusions RECK changes that reduce the ability of the cell to remodel the ECM (overexpression and cell surface shedding) are compensated for by increases in MT1-MMP, and MMP-2 levels as seen by zymography.

Background: MT1-MMP is a cell-surface enzyme whose regulation of pro-MMP-2 and ERK activation position it as a key facilitator of ECM remodelling and cell migration. These processes are modulated by endogenous MMP inhibitors, such as RECK, a GPI-anchored protein which has been shown to inhibit both MT1-MMP and MMP-2 activity. Our previous studies have revealed a link between MT1-MMP levels, and pro-MMP-2 and ERK activation in mammalian cells, as well as MT1-MMP and RECK co-localization in Xenopus embryos. We here investigated how modulation of RECK would impact MT1-MMP and MMP-2 levels, as well as ERK signalling in Xenopus A6 cells. Results: We used a Morpholino approach to knockdown RECK, plasmid transfection to overexpress RECK, and PI-PLC treatment to shed RECK from the cell surface of Xenopus A6 cells. RECK reduction did not alter pERK or MT1-MMP levels, nor MMP-2 activity as measured by zymography; thus RECK-knockdown cells maintained the ability to remodel the ECM. RECK overexpression and PI-PLC treatment both increased ECM remodelling potential through increased MT1-MMP protein and relative MMP-2 activation levels. Conclusions: RECK changes that reduce the ability of the cell to remodel the ECM (overexpression and cell surface shedding) are compensated for by increases in MT1-MMP, and MMP-2 levels as seen by zymography. [ABSTRACT FROM AUTHOR]

Amphibians are regarded as sensitive sentinels of environmental pollution due to their permeable skin and complex life cycle, which usually involves reproduction and development in the aquatic environment. Fungicides are widely applied agrochemicals and have been associated with developmental defects in amphibians; thus, it is important to determine chronic effects of environmentally-relevant concentrations of such contaminants in target cells. Infrared (IR) spectroscopy has been employed to signature the biological effects of environmental contaminants through extracting key features in IR spectra with chemometric methods. Herein, the Xenopus laevis (A6) cell line was exposed to low concentrations of carbendazim (a benzimidazole fungicide) or flusilazole (a triazole fungicide) either singly or as a binary mixture. Cells were then examined using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy coupled with multivariate analysis. Results indicate significant changes in the IR spectra of cells induced by both agents at all concentrations following single exposures, primarily in regions associated with protein and phospholipids. Distinct differences were apparent in the IR spectra of cells exposed to carbendazim and those exposed to flusilazole, suggesting different mechanisms of action. Exposure to binary mixtures of carbendazim and flusilazole also induced significant spectral alterations, again in regions associated with phospholipids and proteins, but also in regions associated with DNA and carbohydrates. Overall these findings demonstrate that IR spectroscopy is a sensitive technique for examining the effects of environmentally-relevant levels of fungicides at the cellular level. The combination of IR spectroscopy with the A6 cell line could serve as a useful model to identify agents that might threaten amphibian health in a rapid and high throughput manner. [ABSTRACT FROM AUTHOR]

Abstract: We have developed a new method of controlling the pipette for scanning ion conductance microscopy to obtain high-resolution images faster. The method keeps the pipette close to the surface during a single line scan but does not follow the exact surface topography, which is calculated by using the ion current. Using an FPGA platform we demonstrate this new method on model test samples and then on live cells. This method will be particularly useful to follow changes occurring on relatively flat regions of the cell surface at high spatial and temporal resolutions. [Copyright &y& Elsevier]

Abstract: Heat shock proteins (HSPs) are molecular chaperones that are involved in protein folding and translocation. During heat shock, both constitutive and stress-inducible HSPs bind to and inhibit irreversible aggregation of denatured protein and facilitate their refolding once normal cellular conditions are re-established. Recent interest in HSPs has been propelled by their association with various human diseases. Amphibian model systems, as shown in this review, have had a significant impact on our understanding of hsp gene expression and function. Some amphibian hsp genes are expressed constitutively during oogenesis and embryogenesis, while others are developmentally regulated and enriched in selected tissues in a stress-inducible fashion. For example, while hsp70 genes are heat-inducible after the midblastula stage, hsp30 genes are not inducible until late neurula/early tailbud. This particular phenomenon is likely controlled by chromatin structure. Also, hsp genes are expressed during regeneration, primarily in response to wounding-associated trauma. The availability of amphibian cultured cells has enabled the analysis of hsp gene expression induced by different stresses (e.g. cadmium, arsenite, proteasome inhibitors etc.), HSP intracellular localization, and their involvement in stress resistance. Furthermore, hyperthermia treatment of adult amphibians reveals that certain tissues were more sensitive than others in terms of hsp gene expression. Finally, this review details the evidence available for the role of amphibian small HSPs as molecular chaperones. [Copyright &y& Elsevier]

Abstract: The effect of hydrostatic pressure on the paracellular ion conductance (Gp) composed of the Na+ conductance (GNa) and the Cl− conductance (GCl) has been Investigated. Gp, GNa and GCl were time-dependently increased after applying an osmotic gradient generated by NaCl with basolateral hypotonicity. Hydrostatic pressure (1–4cm H2O) applied from the basolateral side enhanced the osmotic gradient-induced increase in Gp, GNa and GCl in a magnitude-dependent manner, while the hydrostatic pressure applied from the apical side diminished the osmotic gradient-induced increase in Gp, GNa and GCl. How the hydrostatic pressure influences Gp, GNa and GCl under an isosmotic condition was also investigated. Gp, GNa and GCl were stably constant under a condition with basolateral application of sucrose canceling the NaCl-generated osmotic gradient (an isotonic condition). Even under this stable condition, the basolaterally applied hydrostatic pressure drastically elevated Gp, GNa and GCl, while apically applied hydrostatic pressure had little effect on Gp, GNa or GCl. Taken together, these observations suggest that certain factors controlled by the basolateral osmolality and the basolaterally applied hydrostatic pressure mainly regulate the Gp, GNa and GCl. [Copyright &y& Elsevier]

To determine whether ceramide mediates regulation of the renal epithelial sodium channel (ENaC) by tumor necrosis factor-α (TNF-α), confocal microscopy and patch- clamp experiments were performed in A6 distal nephron cells. We found that TNF-α (100 ng/ml) had no effect on ENaC activity and ceramide level when the cells were grown in the presence of aldosterone, but significantly inhibited ENaC and induced ceramide production after the cells were pretreated with LY 294002, an inhibitor of 1 phosphatidylinositol 3-kinase, for 24 h. The inhibition of ENaC induced by TNF-α was mimicked by exogenous sphingomyelinase (0.1 U/mI) and C2-ceramide (50 μM), but neither C2-dihydroceramide, a membrane-impermeable analog of C2-ceramide, nor choline, and abolished by pretreatment with GF109203X, a protein kinase C (PKC) inhibitor. C2-ceramide failed to affect ENaC in the cells pretreated with GF109203X, but not in the cells pretreated with PD-98059, a mitogen-activated protein kinase kinase inhibitor. C2- ceramide induced the externalization of phosphatidylserine (PS) in control A6 cells, but not in the cells pretreated with GF109203X. Together with our previous finding that cytosolic PS maintains ENaC activity in A6 cells, these data suggest that ceramide mediates TNF-α inhibition of the renal ENaC via a pathway associated with PKC- 1 dependent externalization of PS. [ABSTRACT FROM AUTHOR]

Activation of epithelial sodium channels (ENaC) by aldosterone, insulin, or insulin-1ike growth factor-1 (IGF-1) in renal epithelial cells (including the Xenopus Iaevis renal cell line A6) appears to share some common signaling elements subsequent to the initial insulin or IGF-1 receptor activation. Previously, the convergence point for insulin or IGF-1 and aldosterone signaling was assumed to be downstream of the receptor at the level of phosphatidylinositol 3-kinase (PI3-K); however, this study shows aldosterone directly transactivates the IGF-1 receptor (IGF-1R). In A6 cells, 10-min exposure to aldosterone increased the phosphorylation of the IGF-1 receptor, insulin receptor substrate-1 (IRS-1), and Akt (PKB). Furthermore, aldosterone activated PI3-K and phosphorylation of the most downstream element, Akt, was blocked by the specific PI3-K inhibitor LY-294002. Transactivation requires aldosterone binding to the mineralocorticoid/glucocorticoid receptor and does not require transcription. [ABSTRACT FROM AUTHOR]

Amiloride-sensitive epithelial Na+ channels (ENaC) play a crucial role in Na+ transport and fluid reabsorption in the kidney, lung, and colon. The magnitude of ENaC-mediated Na+ transport in epithelial cells depends on the average open probability of the channels and the number of channels on the apical surface of epithelial cells. The number of channels in the apical membrane, in turn, depends on a balance between the rate of ENaC insertion and the rate of removal from the apical membrane. ENaC is made up of three homologous subunits: á, β, and γ. The COOH-terminal domain of all three subunits is intracellular and contains a proline-rich motif (PPxY). Mutations or deletion of this PPxY motif in the β- and γ-subunits prevent the binding of one isoform of a specific ubiquitin ligase, neural precursor cell-expressed, developmentally downregulated protein (Nedd4-2), to the channel in vitro and in transfected cell systems, thereby impeding ubiquitin conjugation of the channel subunits. Ubiquitin conjugation would seem to imply that ENaC turnover is determined by the ubiquitin-proteasome system, but when Madin-Darby canine kidney cells are transfected with ENaC, ubiquitin conjugation apparently leads to lysosomal degradation. However, in untransfected renal cells (A6) expressing endogenous ENaC, ENaC is indeed degraded by the ubiquitin-proteasome system. Nonetheless, in both transfected and untransfected cells, the rate of ENaC degradation is apparently controlled by Nedd4-2 activity. In this review, we discuss the role of the ubiquitin conjugation and the aitemative degradative pathways (lysosomal or proteasomal) in regulating the rate of ENaC turnover in untransfected renal cells and compare this regulation to that of transfected cell systems. [ABSTRACT FROM AUTHOR]

Serum- and glucocorticoid-induced kinase 1 (SGKI) is thought to be an important regulator of Na+ reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na+ transport. Previous studies have shown that SGK1 increases Na+ transport and epithelial Na+ channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na+-K+-ATPase activity, the transporter responsible for basolateral Na+ efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na+-K+-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1S425DT) increased the transport activity of Na+-K+-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na+-K+-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na+ pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1S425DT, induced an ∼2.5-fold increase in total protein and plasma membrane Na+-K+-ATPase a α1-subunit abundance. We conclude that aldosterone increases the abundance of Na+-K+-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli. [ABSTRACT FROM AUTHOR]

Reabsorption of sodium by the epithelial sodium channel (ENaC) is essential for maintaining the volume of the extracellular compartment and blood pressure. The function of ENaC is regulated primarily by aldosterone, antidiuretic hormone [arginine vasopressin (AVP)], and insulin, but the molecular mechanisms that increase channel activity are still poorly understood. It has been proposed that the related serine/threonine kinases serum- and glucocorticoid-induced kinase (Sgk1) and protein kinase B (Akt) mediate activation of ENaC. Here, we addressed the question of whether there is functional specificity of these kinases for the activation of ENaC in epithelial cells of the distal renal tubule. We demonstrate that Akt does not increase ENaC function under basal conditions or after stimulation with aldosterone, insulin, or AVP. In contrast, under the same experimental conditions, Sgkl1increases ENaC activity by 10-fold. The effect of Sgk1 is additive to that of aldosterone, whereas, in the presence of active Sgk1, cells do not further respond to insulin or AVP. We conclude that, in cells expressing both kinases, modulation of ENaC activity is mediated by Sgk1 but not by Akt1. [ABSTRACT FROM AUTHOR]

Abstract: Here, we report changes gene expression and morphology of the renal epithelial cell line, A6, which was derived from Xenopus laevis adult kidney that had been induced by long-term culturing with a three-dimensional clinostat. An oligo microarray analysis on the A6 cells showed that mRNA levels for 52 out of 8091 genes were significantly altered in response to clinorotation. On day 5, there was no dramatic change in expression level, but by day 8 and day 10, either upregulation or downregulation of gene expression became evident. By day 15, the expression levels of 18 out of 52 genes had returned to the original levels, while the remaining 34 genes maintained the altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in the mRNA levels of selected genes were found only under clinorotation and not under hypergravity (7g) or ground control. Morphological changes including loss of dome-like structures and disorganization of both E-cadherin adherence junctions and cortical actin were also observed after 10 days of culturing with clinorotation. These results revealed that the expression of selected genes was altered specifically in A6 cells cultured under clinorotation. [Copyright &y& Elsevier]

We present a comparative study in frog urinary bladders (FUB) and A6 cell monolayers (A6CM) on the effect of La3+ on tight junction (TJ) dynamics. These tissues react similarly to changes of basolateral Ca2+ (Ca2+bl), while responding differently to the action of La3+bl. In FUB, La3+bl shows a Ca2+-antagonistic effect that promotes TJ opening in the presence of a normal Ca2+bl concentration. In A6CM, in contrast, La3+bl always shows a clear Ca2+-agonistic effect. The fact that a concentration of La3+bl one fifth of the normal Ca2+bl leads in FUB to TJ opening and in A6CM to a complete recovery of the TJ seal indicates a high affinity of La3+ for the Ca2+-binding sites in both tissues. In FUB, apical La3+ (La3+ap) exhibits, differently from its basolateral effect, an evident Ca2+-agonistic effect, suggesting a dual effect of La3+, depending on which side of the bladder La3+ is applied. In A6CM La3+ap has a Ca2+-agonistic effect similar to La3+bl. The effects of La3+bl in FUB and in A6CM are consistent, according to our previous publications, with La3+ acting antagonistically or agonistically, respectively, on the Ca2+ binding sites ofzonula adhaerens. Despite the fact that the effect of La3+ap is clear in both tissues, its site of action is yet to be determined. Protonation of the Ca2+-binding sites causes a decrease of its agonistic effect on A6CM, consistent with a negatively charged binding site. In A6CM La3+ apparently replaces Ca2+, mimicking the effect of Ca2+ triggering the cascade of events leading to TJ closure. In FUB, La3+ interacts with the binding sites, dislodging Ca2+, with a high affinity, but this interaction is inadequate to initiate or sustain the process of junction closing. Possibly, the difference between the two preparations resides in subtle conformation differences of the outer segment of E-cadherin molecules. [ABSTRACT FROM AUTHOR]

The epithelial sodium channel is found in apical membranes of a variety of native epithelial tissues, where it regulates sodium and fluid balance. In vivo, a number of hormones and other endogenous factors, including polyunsaturated fatty acids (PUFAs), regulate these channels. We tested the effects of essential n-3 and n-6 PUFAs on amiloride-sensitive sodium transport in A6 epithelial cells. Eicosapentaenoic acid [EPA; C20:5(n-3)] transiently stimulated amiloride-sensitive open-circuit current (INa) from 4.0 ± 0.3 to 7.7 ± 0.3 µA/cm² within 30 min (P < 0.001). No activation was seen in the presence of 10 µM amiloride. In cell-attached but not in cell-excised patches, EPA acutely increased the open probability of sodium channels from 0.45 ± 0.08 to 0.63 ± 0.10 (P = 0.02, paired t-test), n-6 PUFAs, including linoleic acid (C18:2), eicosatetraynoic acid (C20: 4), and docosapentanoic acid (C22:5) had no effect, whereas n-3 docosahexanoic acid (C22:6) activated amiloride-sensitive INa in a manner similar to EPA. Activation of INa by EPA was prevented by H-89, a PKA inhibitor. Similarly, PKA activity was stimulated by EPA. Nonspecific stimulation of phosphodiesterase activity by COCl2 completely prevented the effect of EPA on sodium transport. We conclude that n-3 PUFAs activate epithelial sodium channels downstream of cAMP in a cAMP-dependent pathway also involving PKA. [ABSTRACT FROM AUTHOR]

Background: Continuous high spatial resolution observations of living A6 cells would greatly aid the elucidation of the relationship between structure and function and facilitate the study of major physiological processes such as the mechanism of action of aldosterone. Unfortunately, observing the micro-structural and functional changes in the membrane of living cells is still a formidable challenge for a microscopist. Method: Scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe, has been shown to be suitable for imaging non-conducting surfaces bathed in electrolytes. A specialized version of this microscopy has been developed by our group and has been applied to image live cells at high-resolution for the first time. This method can also be used in conjunction with patch clamping to study both anatomy and function and identify ion channels in single cells. Results: This new microscopy provides high-resolution images of living renal cells which are comparable with those obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Continuous 24 h observations under normal physiological conditions showed how A6 kidney epithelial cells changed their height, volume, and reshaped their borders. The changes in cell area correlated with the density of microvilli on the surface. Surface microvilli density ranged from 0.5 μm-2 for extended cells to 2.5 μm2 for shrunk cells. Patch clamping of individual cells enabled anatomy and function to be correlated. Conclusions: Scanning ion conductance microscopy provides unique information about living cells that helps to understand cellular function. It has the potential to become a powerful tool for research on living renal cells. [Copyright &y& Elsevier]

In cultured A6 monolayers from distal Xenopus kidney, external Ni2+ stimulated active Na+ uptake via the epithelial Na+ channel, ENaC. Transepithelial capacitance measurements ruled out exocytosis of ENaC-containing vesicles underlying the Ni2+ effect. Na+ current noise analysis was performed using the neutral Na(+) -channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide (CDPC) and amiloride. The analysis of CDPC-induced noise in terms of a three-state channel model revealed that Ni2+ elicits an increase in the number of open channels as well as in the spontaneous open probability. While Ni2+ had no influence on CDPC-blocker kinetics, the macroscopic and microscopic blocking kinetics of amiloride were affected. Ni2+ turned out to compete with amiloride for a putative binding site but not with CDPC. Moreover, external Na(+)--known to compete with amiloride and so producing the "self-inhibition" phenomenon--and Ni2+ exerted mutually exclusive analogous effects on amiloride kinetics. Na+ current kinetics revealed that Ni2+ prevents ENaC to be downregulated by self-inhibition. Co2+ behaved similarly to Ni2+, whereas Zn2+ did not. Attempts to disclose the chemical nature of the site reacting with Ni2+ suggested cysteine but not histidine as reaction partner. [ABSTRACT FROM AUTHOR]

As potential autocrine or paracrine factors, extracellular nucleotides are known to be important regulators of renal ion transporters by activating cell surface receptors and intracellular signaling pathways. We investigated the influence of extracellular adenine nucleotides on Na+/H+ exchanger isoform 3 (NHE3) activity in A6-NHE3 cells. This is a polarized cell line obtained by stable transfection of A6 cells with the cDNA encoding the rat isoform of NHE3, which is expressed on the apical membrane. Basolateral addition of the P2Y1 agonist, 2-MeSADP, induced an inhibition of NHE3 activity, which was prevented by preincubation with selective P2Y1 antagonists, MRS 2179 (N6-methyl-2?-deoxyadenosine-3?,5?-bisphosphate) and MRS 2286 (2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane-1,3-bisoxy(diammoniumphosphate)). NHE3 activity was also significantly inhibited by ATP and ATP-g-S but not by UTP. 2-MeSADP induced a P2Y1 antagonist-sensitive increase in both [Ca2+]i and cAMP production. Pre-incubation with a PKC inhibitor, Calphostin C, or the calcium chelator BAPTA-AM, had no effect on the 2-MeSADP-dependent inhibition of NHE3 activity, whereas this inhibition was reversed by either incubation with the PKA inhibitor H89 or by mutation of two PKA target serines (S552 and S605) on NHE3. Pre-incubation of the A6-NHE3 cells with the synthetic peptide, Ht31, which prevents the binding between AKAPs and the regulatory PKA subunits RII, also prevented the 2-MeSADP-induced inhibition of NHE3. We conclude that only the cAMP/PKA pathway is involved in the inhibition of NHE3 activity. [ABSTRACT FROM AUTHOR]

Confluent high-density cell cultures of A6 cells derived from adult male Xenopus kidney exhibit spontaneous dome-formation at 1 g. To determine whether this morphogenetic property is altered by gravity, we used a three-dimensional (3D) clinostat to subject the cells to simulated microgravity, and a centrifuge to subject them to hypergravity. We used the generation orbit control method as the new rotation control system of the 3D-clinostat, not the random method. The growth of A6 cells was significantly enhanced by hypergravity, but significantly reduced by simulated microgravity. Dome promoted dome formation and induced tubule morphogenesis, compared to the control at 1 g. These results indicated that changes in gravity influence the morphogenetic properties of A6 cells, such as dome formation and tubule morphogenesis. When dome formation by A6 cells at high, confluence was induced spontaneously in the control 1 g culture, the gene expression of the HGF family of pleiotropic factors, such as HGF-like protein, (HLP) and growth factor-Livertine (GF-Livertine), an epithelial serine protease of channel activating protease 1 (CAP1), and Na+, K+-adenosine triphosphatase (ATPase), increased. Simulated microgravity increased the gene expression of activin A and reduced the gene expression of HLP, GF-Livertine, CAP1, and, Na+, K+-ATPase. Hypergravity, on the other hand, decreased the gene expression of activin A and increased the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. These results suggest that the effects of gravitational changes on expression of the HGF family member gene, CAP1, and Na+, K+-ATPase gene may be important for the cell growth, tubule morphogenesis and dome formation of A6 cells in altered gravity. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that in A6 renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A1 and A2A receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A3 receptor-selective agonist, 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca2+ influx. The agonist-induced rise in [Ca2+]i was significantly inhibited by the selective adenosine A3 receptor antagonists, 2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A1 or A2 receptors supporting the hypothesis that Cl-IB-MECA increases [Ca2+]i by interacting exclusively with A3 receptors. Cl-IB-MECA-elicited Ca2+ entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca2+]i response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A3 receptor antagonist. Altogether, these data suggest that in A6 cells a Gs/protein kinase A pathway is involved in the A3 receptor-dependent increase in calcium entry. [ABSTRACT FROM AUTHOR]

Role of SGK in mineralocorticoid-regulated sodium transport. Mineralocorticoids stimulate electrogenic Na+ transport in tight epithelia by altering the transcription of specific genes. Although the earliest mineralocorticoid effect is to increase the activity of the epithelial sodium channel (ENaC), ENaC mRNA and protein levels do not change. Instead, physiologic observations suggest that a mineralocorticoid target gene(s) encodes an ENaC regulator(s). To begin to identify and characterize mineralocorticoid-regulated target genes, we used suppression-subtractive hybridization to generate a cDNA library from A6 cells, a stable cell line of Xenopus laevis of distal nephron origin. A serine-threonine kinase, SGK, was identified from this screen. Sequence comparison revealed that frog, rat, and human SGK are 92% identical and 96% similar at the amino acid level. SGK mRNA was confirmed by Northern blot to be strongly and rapidly corticosteroid stimulated in A6 cells. In situ hybridization revealed that SGK was strongly stimulated by aldosterone in rat collecting duct but not proximal tubule cells. Low levels of SGK were present in rat glomeruli, but SGK was unregulated in this structure. Finally, SGK stimulated ENaC activity approximately sevenfold when coexpressed in Xenopus laevis oocytes. These data suggest that SGK is an important mediator of aldosterone effects on Na+ transport in tight epithelia. In view of the existence of SGK homologues in invertebrates, it is interesting to speculate that SGK is an ancient kinase that was adapted to the control of epithelial Na+ transport by early vertebrates as they made the transition from a marine to a freshwater environment. [ABSTRACT FROM AUTHOR]

Measurement of cAMP-dependent protein kinase activity using a fluorescent-labeled Kemptide. Background . Traditional protein kinase assays include the use of [32 P] labeled ATP as phosphate donor and a substrate protein or peptide as phosphoreceptor. Since this approach has a number of drawbacks in addition to generating ionizing radiation, several non-isotopic methods have been developed. Although shown to reflect the activity of purified enzymes, none have been demonstrated to detect physiological changes in endogenous enzyme activity in cell homogenates. Methods . Studies were performed to examine the kinetics, reproducibility, and optimal assay conditions of a novel non-radioisotopic kinase assay that detects PKA activity by phosphorylation of the peptide substrate Kemptide covalently bound to a fluorescent molecule (f-Kemptide). Basal and agonist-induced PKA activity in epithelial cell homogenates was measured. Results . The kinetics of f-Kemptide were similar to the standard radioisotopic method with intraassay and interassay variations of 5.6 ± 0.8% and 14.3 ± 2.6%, respectively. Neither fluorescence quenching nor enhancing effects were found with consistent amounts of homogenate protein. Specific PKA activity was determined as the IP20 -inhibitable fraction to account for nonspecific phosphorylation, perhaps due to S6 kinase or a similar enzyme. The basal activity of 38% of total PKA in A6 cells increased by 84% after exposure to vasopressin and by 58% after short exposure to forskolin. In T84 cells exposed to VIP there was a 360% increase over basal activity. Conclusions . These results show that f-Kemptide exhibits acceptable kinetics, and that the assay system can quantitatively and reproducibly measure basal and stimulated PKA activity in cell homogenates. [ABSTRACT FROM AUTHOR]

The presence of a Na/Ca exchanger in A6 cells was investigated by measuring intracellular calcium (Ca) fluctuations and the Ca fluxes through the basolateral membranes (blm) of the cell monolayer. Removal of Na from the medium produced a transient increase in Ca followed by a regulatory phase returning Ca to control levels in 3-4 min, this phase being greatly accelerated (< 60 s) by NaCl addition (apparent K of approximately 5 mM Na). The Ca increase was only found with the Na-free medium on the basolateral side of the cell monolayer. A twofold increase in the Ca influx was observed under these conditions. In Ca- depleted cells, the initial Ca increase after Ca addition to the medium was greater when the putative Na/Ca exchanger was not functioning (i.e. in a Na-free medium). Ca effluxes through the blm of the monolayer were greatly and transiently increased by a Na-free medium on the serosal side and blocked by orthovanadate (1 mM). The Ca increase induced by a hypo-osmotic shock was greater in cells bathed in a Na-medium, conditions expected to block the activity of the Na/Ca exchanger. These findings support the hypothesis that a Na/Ca exchanger is present on the blm of A6 cells and affirm its role in Ca homeostasis in steady-state conditions and following osmotic shock. In addition, a Ca pump also located on the blm and Ca stores sensitive to inositol 1,4,5-trisphosphate were found to be implicated in Ca homeostasis. [ABSTRACT FROM AUTHOR]

We report the synthesis of a radioactive, methylated analog of bromoamiloride which inhibits the amiloride-sensitive, epithelial Na+ channel reversibly and with high affinity. This synthesis was achieved by methylation of a nitrogen in the acylguanidinium moiety with tritiated methyliodide of high specific activity. This methylated bromoamiloride molecule (CH3BrA) was purified by both thin layer and high performance liquid chromatography. Proton nuclear magnetic resonance and mass spectroscopy techniques were used to determine the structure of this analog. This compound inhibited both short-circuit current of in vitro frog skin and 22Na+ influx into apical plasma membrane vesicles made from cultured toad kidney cells (line A6) with the same or lower apparent inhibitory dissociation constant as bromoamiloride. Irradiation with ultraviolet light rendered this inhibition irreversible in both A6 vesicles and frog skin. Preparation of radioactive CH3BrA yielded specific activities in excess of 1 Ci/mmol. We suggest that this compound will be useful in the isolation and purification of this ubiquitous Na+ channel. [ABSTRACT FROM AUTHOR]

The apical membrane of epithelial cells from the A6 cell line grown on impermeable substrata was studied using the patch-clamp technique. We defined the apical membrane as that membrane in contact with the growth medium. In about 50% of the patches, channels with single-unit conductances of 360 +/- 45 pS in symmetrical 105 mM NaCl solutions, and characteristic voltage-dependent inactivation were observed. Using excised membrane patches and varying the ionic composition of the bathing medium, we determined that the channels were anion selective, with a permeability ratio for Cl- over Na+ of about 9:1, calculated from the reversal potential using the constant-field equation. The channel was most active at membrane potentials between +/- 20 mV and inactivated, usually within a few seconds, at higher potentials of either polarity. Reactivation from this inactivation was slow, sometimes requiring minutes. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to one or more submaximal conductance levels. The channel was inhibited by the disulfonic stilbene SITS in a manner characteristic of reversible open-channel blockers. [ABSTRACT FROM AUTHOR]

To assess the role of chloride in cell volume and sodium transport regulation, we measured cell height changes (CH), transepithelial chloride and sodium fluxes, and intracellular chloride content during challenge with hyposmotic solutions under open circuit (OC) conditions. CH maximally increased following hyposmotic challenge within approximately 5 minutes. The change in CH was smaller under short circuit (SC) conditions or following replacement of chloride in the mucosal solution by gluconate or cyclamate (Cl(-)-freem). When corrected for the osmotically inactive cell volume (30 +/- 2%), delta CH for controls (OC) were greater than predicted for an ideal osmometer. In contrast, delta CH for Cl(-)-freem or SC conditions were similar to that predicted for an ideal osmometer. Na+ and Cl- mucosa-to-serosa fluxes increased following hyposmotic challenge. Chloride fluxes increased maximally within 5 min, then decreased. In contrast, the Na+ flux increased slowly and reached a steady state after approximately 25 min. Under isosmotic conditions, exposure to Cl(-)-freem solutions led to decreases in the transepithelial conductance, Na+ flux, and CH. Chloride permeabilities in the apical and basolateral membranes were detected using the fluorescent intracellular chloride indicator MQAE. The results indicate that during osmotic swelling, the entry of both sodium and chloride is increased. The time courses of these increases differ, suggesting distinct mechanisms for the osmotic regulation of these apical membrane transport processes. [ABSTRACT FROM AUTHOR]

The labeling of specific cell surface proteins with biotin was used to examine both protein distribution and delivery of newly synthesized proteins to the apical and basolateral cell surface in A6 cells. Steady-state metabolic labeling with [S]methionine followed by specific cell surface biotinylation demonstrated polarization of membrane proteins. The delivery of newly synthesized proteins to the apical or basolateral cell surface was examined by metabolic labeling with [S]methionine using a pulse-chase protocol in combination with specific cell surface biotinylation. Newly synthesized biotinylated proteins at the apical cell surface reached a maximum after a 5 min chase, and then fell over the remainder of a 2 hr chase. The bulk flow of newly synthesized proteins to the basolateral membrane slowly rose to a maximum after 90 min. The detergent Triton X-114 was used to examine delivery of hydrophilic and hydrophobic proteins to the cell surface. Delivery of both hydrophilic and hydrophobic proteins to the apical cell surface reached a maximum 5 to 10 min into the chase period. The arrival of hydrophilic proteins at the basolateral surface showed early delivery and a maximum peak delivery at 120 min into the chase period. In contrast, only an early peak of delivery of newly synthesized hydrophobic proteins to the basolateral membrane was observed. [ABSTRACT FROM AUTHOR]

K channels sensitive to intracellular ATP (K channels) have been described in a number of cell types and are selectively inhibited by sulfonylurea drugs. To look for the presence of this type of K channel in the basolateral membrane of tight epithelia, we have used an amphibian renal cell line, the A6 cells, grown on filters. After the selective permeabilization of the apical membrane with amphotericin B, the basolateral conductance was studied under voltage-clamp conditions. Tolbutamide inhibited 65.8 ± 6.3% of the barium-sensitive current. The tolbutamide-sensitive conductance had an equilibrium potential of −83 ± 1 mV and was inward rectifying in spite of the outwardly directed K gradient. Similar results were obtained with glibenclamide. The half-inhibition constants were 25.7 ± 3.0 μ m and 0.114 ± 0.018 μ m for tolbutamide and glibenclamide respectively. To study the relation between cellular ATP and the activity of this conductance, A6 cells were treated with glucose (5 m m) and insulin (250 μU/ml). This maneuver significantly increased the cellular ATP and abolished the tolbutamide-sensitive conductance. A sulfonylurea-sensitive K conductance is present and active in the basolateral membrane of A6 cells. This conductance appears to be modulated by physiological changes of intracellular ATP. [ABSTRACT FROM AUTHOR]

The polarity of cell-surface membrane movements and their regulation by adrenal steroid hormones (10 m aldosterone) and vasopressin or vasotocin were studied in A6 cells. This cell line is derived from the Xenopus laevis distal nephron and displays regulated Na reabsorption but is devoid of regulated water transport. Apical and basolateral membrane movements and their hormonal regulation were characterized by measuring the uptake of the fluid phase marker horseradish peroxidase (HRP) and the secretion of proteins on both sides of cell monolayers cultured on filters. The intracellular accumulation of HRP was visualized by electron microscopy and quantified by the measure of cell-associated peroxidase activity. The rate of intracellular HRP accumulation corresponded to 0.01 nl/minute/filter (4.7 cm) from the apical side and was 20-32 times faster from the basolateral side. In contrast, the level of protein secretion was 3.5 times higher apically than basolaterally. Among the secreted proteins some were found to be secreted essentially apically, and others basolaterally. Vasotocin increased apical endocytosis (1.88-fold) and apical protein secretion (1.49-fold) in cells pretreated with aldosterone. Basolaterally, only the endocytosis was increased, and to a smaller extent (1.36-fold). These effects of vasotocin depended on aldosterone pretreatment and could be mimicked with forskolin and 8-bromoadenosine 3′∶5′-cyclic monophosphate (BrcAMP). Measurements of intracellular cAMP levels showed that there was a rankorder correlation between the induced level of intracellular cAMP and that of apical endocytosis. This study shows that vasotocin has a polarized stimulatory action on apical endocytosis and protein secretion in A6 cells, and that the mediation of this action by cAMP is aldosterone dependent. [ABSTRACT FROM AUTHOR]

To study the properties of the basolateral membrane conductance of an amphibian epithelial cell line, we have adapted the technique of apical membrane selective permeabilization (Wills, N.K., Lewis, S.A., Eaton, D.C. 1979b, J. Membrane Biol. 45:81-108). Monolayers of A6 cells cultured on permeable supports were exposed to amphotericin B. The apical membrane was effectively permeabilized, while the high electrical resistance of the tight junctions and the ionic selectivity of the basolateral membrane were preserved. Thus the transepithelial current-voltage relation reflected mostly the properties of the basolateral membrane. Under "basal" conditions, the basolateral membrane conductance was inward rectifying, highly sensitive to barium but not to quinidine. After the induction of cell swelling either by adding chloride to the apical solution or by lowering the osmolarity of the basolateral solution, a large outward-rectifying K+ conductance was observed, and addition of barium or quinidine to the basolateral side inhibited, respectively, 82.4 +/- 1.9% and 90.9 +/- 1.0% of the transepithelial current at 0 mV. Barium block was voltage dependent; the half-inhibition constant (Ki) varied from 1499 +/- 97 microM at 0 mV to 5.7 +/- 0.5 microM at -120 mV. Cell swelling induces a large quinidine-sensitive K+ conductance, changing the inward-rectifying basolateral membrane conductance observed under "basal" conditions into a conductance with outward-rectifying properties. [ABSTRACT FROM AUTHOR]

Aldosterone increases the basolateral conductance in target epithelia. The basolateral membrane of tight epithelia contains two different types of K conductances (G), a 'resting' and a volume-activated G. We have studied the early effects (at 4 hours) of 500 nmol/l aldosterone on the basolateral membrane G of A6 cells (a Xenopus laevis kidney cell line), after the permeabilization of the apical membrane with amphotericin B. In the presence of a 97 to 3 mmol/l apical to basolateral K gradient, the 'resting', inward rectifying G was similar in control and aldosterone treated cells. In contrast, aldosterone induced a 2-fold increase of the volume-activated quinidine sensitive G. [ABSTRACT FROM AUTHOR]

Quantitative real-time polymerase chain reaction is a widely used technique that relies on reference genes for the normalisation of gene expression. These reference genes are constitutively expressed and must remain stable across all samples and treatments. Stability of housekeeping genes may vary and must be optimised for a specific tissue, sample or cell line. Here we present a study screening for possible reference gene candidates, eef1a1 , rpl8 , sub1.L , clta , H4 and odc1 , in the Xenopus laevis (A6) kidney cell line. Quantification cycle results were analysed using geNorm to calculate the average expression stability and the coefficient of variation (CV) for each candidate reference gene. All of the tested genes met the guidelines for stable reference genes, namely an average expression stability of < 0.5 and a CV value of < 0.2, with eef1a1 > sub1.L > rpl8 > clta > odc1 > H4 . By using pairwise variation analysis, the optimal number of reference targets was determined to be 2. As such, we report that the reference genes eef1a1 and sub1.L should be used to achieve optimal normalisation in A6 cells.