Epithelial A549 Research Articles

Insulin increases sodium (Na+) channel density in A6 epithelia: implications for expression of hypertension.

Essential or primary hypertension is a multifactorial disease that is expressed as a result of complex interactions between genes and environmental influences. Several mutations in many different proteins are associated with expression of hypertension, including abnormalities in the epithelial sodium channel (ENaC) found in absorptive organs (i.e., distal colon, distal tubule of the nephron). Some of these mutations result in structural and/or functional alterations in ENaC-mediated Na+ entry in epithelia responsible for fluid and electrolyte balance and are associated with expression of hypertension. Studies support the notion that there is a link between ENaC and hypertension of both the monogenic (single gene mutation) and primary or essential type (a multifactorial disease). Alterations of other aspects of the environment of absorptive cells (e.g., hyperinsulinemia, hyperaldosteronemia, high plasma cortisol, high plasma Na+) have also been shown to elicit hyperabsorption of Na+ via ENaC and therefore could contribute significantly to expression of hypertension in people with intermediate phenotypes. This article describes an initial study in which the effects of an environmental factor, extracellular levels of insulin, on ENaC were examined in a normal kidney cell model. Electrophysiologic techniques revealed that ENaC density rapidly increased in response to addition of insulin to the basolateral bath. This autoregulatory recruitment of Na+ total channel density masked a slight decrease in open channel probability. Insulin's effect on ENaC function and implications on fluid and electrolyte balance and expression of primary hypertension is discussed.

Aldosterone action: induction of p21(ras) and fra-2 and transcription-independent decrease in myc, jun, and fos.

Adrenal steroids induce an increase in transcellular Na+ reabsorption across Xenopus laevis A6 cell epithelia that requires the action of transcriptionally regulated gene products. In a previous study we identified K-ras2 as an aldosterone-upregulated mRNA in A6 epithelia. Here, we show that in vivo injection of aldosterone in Xenopus (2.5 h) increases K-ras2 mRNA specifically in the kidney (2.5-fold) and that in A6 epithelia aldosterone (2.5 h) increases Ras protein synthesis ( approximately 6-fold). Xl-ras, another ras mRNA expressed at a low level in A6 cells, was also induced (2-fold). Aldosterone was shown to regulate the mRNA levels of several transcription factors as well. After 2 h of aldosterone treatment, fra-2 mRNA was upregulated by 130%, whereas c-myc, c-jun, c-fos, and glucocorticoid receptor mRNAs were downregulated by 23-43%. After 16 h, c-fos and GR mRNAs were further decreased, whereas levels of fra-2, c-jun, and c-myc began to return to control levels. Interestingly, the downregulation of the protooncogene mRNAs was independent of transcription. These results support the view that aldosterone exerts complex pleiotropic transcriptional and nontranscriptional actions that involve the regulation of signaling cascade elements (i.e., K-Ras2) as well as that of transcription factors.

Antiidiotypic Antibody Recognizes an Amiloride Binding Domain within the α Subunit of the Epithelial Na+ Channel

We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na+ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis alphaENaC. RA6.3 specifically recognized an amiloride binding domain within the alpha-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the alpha-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to alphaENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within alphaENaC.

Transepithelial capacitance decrease reveals closure of lateral interspace in A6 epithelia

A sine wave method was used to measure transepithelial capacitance (CT) at 4.1 kHz (CHFT ). Model calculations show that CHFT reflects the equivalent capacitance of the series arrangement of apical and basolateral membrane capacitance. Cell swelling induced by reducing the basolateral osmolality from 260 to 140 mosmol/kg H2O (NaCl or sucrose removal) transiently decreased CHFT. The decrease in CHFT (DeltaCHFT ) reached its maximum 30 s after the onset of cell swelling and a complete recovery of CHFT was attained within 3-4 min. DeltaCHFT could be diminished by manoeuvres that reduced the rate or amplitude of cell swelling, i.e. lowering the temperature or treatment with low concentrations of glutaraldehyde (0.025%). DeltaCHFT increased with the magnitude of the osmotic perturbation but saturated at large volume expansions. DeltaCHFT increased with culture time. Electron micrographs showed a clear correlation between time course of CHFT changes and the closure of the lateral interspace (LIS). A striking correlation between the occurrence of CHFT recovery and the ability of the cells to develop a regulatory volume decrease (RVD) was found: Gd3+ (0.5 mM) inhibited both phenomena. The frequency dependence of CT was obtained from impedance spectra recorded over the range of 4 Hz to 22 kHz. These data agree with model calculations in which the contribution of the access resistance to the lateral membrane was included. All observations are consistent with the idea that DeltaCHFT originates from the closure of the LIS during cell swelling. The latter phenomenon increases the access resistance to the lateral membrane, which results in a marked reduction of the basolateral membrane area detected at high frequencies with capacitance measurements.

Adenosine inhibits the transfected Na+-H+ exchanger NHE3 in Xenopus laevis renal epithelial cells (A6/C1).

1. Adenosine influences the vectorial transport of Na+ and HCO3- across kidney epithelial cells. However, its action on effector proteins, such as the Na+-H+ exchanger NHE3, an epithelial brush border isoform of the Na+-H+ exchanger (NHE) gene family, is not yet defined. 2. The present study was conducted in Xenopus laevis distal nephron A6 epithelia which express both an apical adenosine receptor of the A1 type (coupled to protein kinase C (PKC)) and a basolateral receptor of the A2 type (coupled to protein kinase A (PKA)). The untransfected A6 cell line expresses a single NHE type (XNHE) which is restricted to the basolateral membrane and which is activated by PKA. 3. A6 cell lines were generated which express exogenous rat NHE3. Measurements of side-specific pHi recovery from acid loads in the presence of HOE694 (an inhibitor with differential potency towards individual NHE isoforms) detected an apical resistant Na+-H+ exchange only in transfected cell lines. The sensitivity of the basolateral NHE to HOE694 was unchanged, suggesting that exogenous NHE3 was restricted to the apical membrane. 4. Stimulation of the apical A1 receptor with N 6-cyclopentyladenosine (CPA) inhibited both apical NHE3 and basolateral XNHE. These effects were mimicked by the addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and partially prevented by the PKC inhibitor calphostin C which also blocked the effect of PMA. 5. Stimulation of the basolateral A2 receptor with CPA inhibited apical NHE3 and stimulated basolateral XNHE. These effects were mimicked by 8-bromo-cAMP and partially prevented by the PKA inhibitor H89 which entirely blocked the effect of 8-bromo-cAMP. 6. In conclusion, CPA inhibits rat NHE3 expressed apically in A6 epithelia via both the apical PKC-coupled A1 and the basolateral PKA-coupled A2 adenosine receptors.

Guanine nucleotide binding proteins in cultured renal epithelia: studies with pertussis toxin and aldosterone.

The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPgammaS revealed a class of binding sites with an apparent Kd value of 100 nM and a Bmax of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmax by 50%. The mRNA levels for Galphai-3, Galpha0, Galphas, and Galphaq were not increased after aldosterone. The patterns of small Mr G proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [alpha-32P]GTP, in control membranes, showed either no labeling or a faint band of Mr 59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Gi proteins and stimulates the expression of a specific GTP-binding protein of Mr 59.5 kDa.

Ras pathway activates epithelial Na+ channel and decreases its surface expression in Xenopus oocytes.

The small G protein K-Ras2A is rapidly induced by aldosterone in A6 epithelia. In these Xenopus sodium reabsorbing cells, aldosterone rapidly activates preexisting epithelial Na+ channels (XENaC) via a transcriptionally mediated mechanism. In the Xenopus oocytes expression system, we tested whether the K-Ras2A pathway impacts on XENaC activity by expressing XENaC alone or together with XK-Ras2A rendered constitutively active (XK-Ras2AG12V). As a second control, XENaC-expressing oocytes were treated with progesterone, a sex steroid that induces maturation of the oocytes similarly to activated Ras. Progesterone or XK-Ras2AG12V led to oocyte maturation characterized by a decrease in surface area and endogenous Na+ pump function. In both conditions, the surface expression of exogenous XENaC's was also decreased; however, in comparison with progesterone-treated oocytes, XK-ras2AG12V-coinjected oocytes expressed a fivefold higher XENaC-mediated macroscopic Na+ current that was as high as that of control oocytes. Thus, the Na+ current per surface-expressed XENaC was increased by XK-Ras2AG12V. The chemical driving force for Na+ influx was not changed, suggesting that XK-Ras2AG12V increased the mean activity of XENaCs at the oocyte surface. These observations raise the possibility that XK-Ras2A, which is the first regulatory protein known to be transcriptionally induced by aldosterone, could play a role in the control of XENaC function in aldosterone target cells.

Adhesive but not lateral E-cadherin complexes require calcium and catenins for their formation.

We examined intercadherin interactions in epithelial A-431 cells producing endogenous E-cadherin and recombinant forms of E-cadherin tagged either by myc or by flag epitopes. Three distinct E-cadherin complexes were found. The first is a conventional E-cadherin-catenin complex consisting of one E-cadherin molecule linked either to beta-catenin/alpha-catenin or to plakoglobin/alpha-catenin dimers. The second is a lateral E-cadherin complex incorporating two E-cadherin- catenin conventional complexes combined in parallel fashion via dimerization of the NH2-terminal extracellular domain of E-cadherin. The third complex is likely to contain two E-cadherin-catenin conventional complexes derived from two opposing cells and arranged in an antiparallel fashion. Formation of the antiparallel but not lateral complex strictly depends on extracellular calcium and E-cadherin binding to catenins. Double amino acid substitution Trp156Ala/Val157Gly within the extracellular NH2-terminal E-cadherin domain completely abolished both lateral and antiparallel inter-E-cadherin association. These data support an idea that the antiparallel complex has the adhesion function. Furthermore, they allow us to suggest that antiparallel complexes derive from lateral dimers and this complex process requires catenins and calcium ions.

Swelling-activated cation-selective channels in A6 epithelia are permeable to large cations.

Effects of basolateral monovalent cation replacements (Na+ by Li+, K+, Cs+, methylammonium, and guanidinium) on permeability to 86Rb of volume-sensitive cation channels (VSCC) in the basolateral membrane and on regulatory volume decrease (RVD), elicited by a hyposmotic shock, were studied in A6 epithelia in the absence of apical Na+ uptake. A complete and quick RVD occurred only when the cells were perfused with Na+ or Li+ saline. With both cations, hypotonicity increased basolateral 86Rb release (RblRb), which reached a maximum after 15 min and declined back to control level. When the major cation was K+, Cs+, methylammonium, or guanidinium, the RVD was abolished. Methylammonium induced a biphasic time course of cell thickness (Tc), with an initial decline of Tc followed by a gradual increase. With K+, Cs+, or guanidinium, Tc increased monotonously after the rapid initial rise evoked by the hypotonic challenge. In the presence of K+, Cs+, or methylammonium, RblRb remained high during most of the hypotonic period, whereas with guanidinium blockage of RblRb was initiated after 6 min of hypotonicity, suggesting an intracellular location of the site of action. With all cations, 0.5 mM basolateral Gd3+ completely blocked RVD and fully abolished the RblRb increase induced by the hypotonic shock. The lanthanide also blocked the additional volume increase induced by Cs+, K+, guanidinium, or methylammonium. When pH was lowered from 7. 4 to 6.0, RVD and RblRb were markedly inhibited. This study demonstrates that the VSCCs in the basolateral membrane of A6 cells are permeable to K+, Rb+, Cs+, methylammonium, and guanidinium, whereas a marked inhibitory effect is exerted by Gd3+, protons, and possibly intracellular guanidinium.

Molecular organization of the desmoglein-plakoglobin complex.

Different epithelial intercellular junctions contain distinct complexes incorporating plakoglobin. In adherens junctions, plakoglobin interacts with two molecules, the transmembrane adhesion protein of the cadherin family (e.g. E-cadherin) and alpha-catenin. The latter is thought to anchor the cadherin-plakoglobin complex to the cortical actin cytoskeleton. In desmosomes, plakoglobin forms a complex with desmosomal cadherins, either desmoglein (Dsg) or desmocollin (Dsc), but not with alpha-catenin. To further understand the structure and assembly of the plakoglobin-cadherin complexes we analyzed amino acid residues involved in plakoglobin-Dsg interactions using alanine scanning mutagenesis. Previously, we have shown that plakoglobin interacts with a 72 amino acid-long cytoplasmic domain (C-domain) that is conserved among desmosomal and classic cadherins. In this paper, we show that a row of the large hydrophobic residues located at the C-terminal portion of the Dsg C-domain is indispensable for interaction with plakoglobin. To study a reciprocal site we expressed plakoglobin (MPg) or its mutants tagged by 6 myc epitope in epithelial A-431 cells. Using sucrose gradient centrifugation and subsequent co-immunoprecipitation, MPg was found to be efficiently incorporated into the same type of complexes as endogenous plakoglobin. A major pool of Dsg-plakoglobin complexes sedimented at 8S and exhibited a 1:1 stoichiometry. Using alanine scanning mutagenesis and the co-immunoprecipitation assay we identified nine hydrophobic amino acids within the arm repeats 1-3 of plakoglobin, that are required for binding to Dsg and Dsc. Eight of these amino acids also participate in the interaction with alpha-catenin. No mutations were found to reduce the affinity of plakoglobin binding to E-cadherin. These data provide direct evidence that the same hydrophobic plakoglobin surface is essential for mutually exclusive interaction with distinct proteins such as alpha-catenin and desmosomal cadherins.

Differential effects of phorbol ester (PMA) on blocker-sensitive ENaCs of frog skin and A6 epithelia.

Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) caused complex transient perturbations of amiloride-sensitive short-circuit Na+ currents (INa) in A6 epithelia and frog skins that were tissue and concentration dependent. A noninvasive channel blocker pulse method of noise analysis (18) was used to investigate how PMA caused time-dependent changes of apical membrane epithelial Na+ channel (ENaC) single-channel currents, channel open probabilities (Po), and channel densities (NT). In A6 epithelia, 5 and 50 nM PMA caused within 7 min concentration-dependent sustained decreases of Po (approximately 55% below control, 50 nM) and rapid compensatory transient increases of NT within 7 min ( approximately 220% above control, 50 nM), resulting in either small transient increases of INa at 5 nM PMA or small biphasic decreases of INa at 50 nM PMA. In contrast to A6 epithelia, 50 and 500 nM PMA in frog skin caused after a delay of at least 10 min transient increases of NT to approximately 60-70% above control at 30-60 min. Unlike A6 epithelia, Po was increased approximately 15% above control within 7 min and remained within +/-10-15% of control for the duration of the 2-h experiments. Despite differences in the time courses of secondary inhibition of transport in A6 epithelia and frog skin, the delayed downregulation of transport was due to time-dependent decreases of NT from their preelevated levels in both tissues. Whereas Po is decreased within minutes in A6 epithelia as measured by noise analysis or by patch clamp (8), the discrepancy in regulation of NT in A6 epithelia as measured by noise analysis and patch clamp is most likely explained by the inability of on-cell patches formed before treatment of tissues with PMA to respond to regulation of their channel densities.

Hypotonicity activates a lanthanide-sensitive pathway for K+ release in A6 epithelia.

The nature of the pathway for K+ release activated during regulatory volume decrease (RVD) in A6 epithelia was investigated by measuring cell thickness (Tc) as an index of cell volume and by probing K+ efflux with 86Rb as tracer for K+ (RRb). Cell swelling was induced by sudden reduction of basolateral osmolality (from 260 to 140 mosmol/kgH2O). Experiments were performed in the absence of Na+ transport. Apical RRb was negligible in iso- and hyposmotic conditions. On the other hand, osmotic shock increased basolateral RRb (RblRb) rapidly, reaching a maximum 7 min after the peak in Tc. Quinine (0.5 mM) completely inhibited RVD and RblRb. Also verapamil (0.2 mM) impeded volume recovery considerably; lidocaine (0.2 mM) did not exert a noticeable effect. The K+ channel blocker Ba2+ (30 mM) delayed RVD but could not prevent complete volume recovery. Cs+ inhibited RVD noticeably at concentrations <40 mM. With large Cs+ concentrations (>40 mM), the initial osmometric swelling was followed by a gradual increase of Tc, suggesting activation of Cs+ influx. Chronic exposure of the basolateral surface to 0.5 mM La3+ or Gd3+ completely abolished RVD and RblRb. Acute administration of lanthanides at the time of osmolality decrease did not affect the initial phase of RVD and reduced RblRb only slightly. Apical Gd3+ exerted an inhibitory effect on RVD and RblRb. The effect of Gd3+ should therefore be localized at an intracellular site. The role of Ca2+ entry could be excluded by failure of extracellular Ca2+ removal to inhibit volume recovery. In contrast to lanthanides, chronically and acutely administered Mg2+ (0.5 mM) inhibited RVD and RblRb by approximately 50%. These data suggest that K+ excretion during RVD occurs through a rather poorly selective pathway that does not seem to be directly activated by membrane stretch.

Hormonal regulation of ENaCs: insulin and aldosterone.

Although a variety of hormones and other agents modulate renal Na+ transport acting by way of the epithelial Na+ channel (ENaC), the mode(s), pathways, and their interrelationships in regulation of the channel remain largely unknown. It is likely that several hormones may be present concurrently in vivo, and it is, therefore, important to understand potential interactions among the various regulatory factors as they interact with the Na+ transport pathway to effect modulation of Na+ reabsorption in distal tubules and other native tissues. This study represents specifically a determination of the interaction between two hormones, namely, aldosterone and insulin, which stimulate Na+ transport by entirely different mechanisms. We have used a noninvasive pulse protocol of blocker-induced noise analysis to determine changes in single-channel current (iNa), channel open probability (Po), and functional channel density (NT) of amiloride-sensitive ENaCs at various time points following treatment with insulin for 3 h of unstimulated control and aldosterone-pretreated A6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10-30 min the density of the pool of apical membrane ENaCs (NT) involved in transport. The very early (10 min) increases of channel density were accompanied by relatively small decreases of iNa (10-20%) and decreases of p.o. (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes of iNa, p.o., and NT were transient, returning very slowly over 3 h toward their respective control values at the time of addition of insulin. We conclude that aldosterone and insulin act independently to stimulate apical Na+ entry into the cells of A6 epithelia by increase of channel density.

Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia.

To study and define the early time-dependent response (< or = 6 h) of blocker-sensitive epithelial Na+ channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current (iNa) channel open probability (Po), and channel density (NT) under transient conditions of transport as measured by macroscopic short-circuit currents (Isc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 microA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. NT varied linearly over a 70-fold range of transport (0.5-35 microA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of Po occurred during control (10-20% over 2 h) and aldosterone experimental periods (10-30% over 6 h). When the Po of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, Po was observed to vary inversely with Isc, falling from approximately 0.5 to approximately 0.15 at the highest rates of Na+ transport or approximately 25% per 3-fold increase of transport. Because decreases of Po from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane NT.

Envelope and long terminal repeat sequences of an infectious murine leukemia virus from a human SCLC cell line: implications for gene transfer.

Development of methods for gene transfer into specific cell types or tissues is important for experimental research as well as clinical therapeutical approaches. We report here the cloning and characterization of the envelope (env) gene and the U3 region of a retrovirus from an infected human Small Cell Lung Cancer (SCLC) cell line. The replication of this murine retrovirus is also fully supported by other lung cancer cell lines of different histological origin. We present evidence that a long terminal repeat (LTR)-beta-galactosidase (beta-Gal) reporter construct performed as well as an analogous cytomegalovirus (CMV) promoter beta-Gal construct in the human lung epithelial cell line A549 and in the human larynx carcinoma cell line HEp2.

Steroid hormone-dependent expression of blocker-sensitive ENaCs in apical membranes of A6 epithelia.

Weak channel blocker-induced noise analysis was used to determine the way in which the steroids aldosterone and corticosterone stimulated apical membrane Na+ entry into the cells of tissue-cultured A6 epithelia. Among groups of tissues grown on a variety of substrates, in a variety of growth media, and with cells at passages 73-112, the steroids stimulated both amiloride-sensitive and amiloride-insensitive Na+ transport as measured by short-circuit currents in chambers perfused with either growth medium or a Ringer solution. From baseline rates of blocker-sensitive short-circuit current between 2 and 7 microA/cm2, transport was stimulated about threefold in all groups of experiments. Single channel currents averaged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and were decreased 6-20% from controls by steroid due to the expected decreases of fractional transcellular resistance. Irrespective of baseline transport rates, the steroids in all groups of tissues stimulated transport by increase of the density of blocker-sensitive epithelial Na+ channels (ENaCs). Channel open probability was the same in control and stimulated tissues, averaging approximately 0.3 in all groups of tissues. Accordingly, steroid-mediated increases of open channel density responsible for stimulation of Na+ transport are due to increases of the apical membrane pool of functional channels and not their open probability.

Osmotic regulation of Na+ transport across A6 epithelium: interactions with prostaglandin E2 and cyclic AMP.

Previous work from this laboratory has shown that apical membrane sodium channel activity is stimulated by serosal hyposmotic solutions (Wills, Millinoff & Crowe, 1991). In the present study, we determined whether this stimulation of sodium transport is additive with the actions of prostaglandin E2 (PGE2) or cyclic AMP (cAMP). Addition of exogenous PGE2 (100 nM; serosal bath) to isosmotic solutions led to large increases in the amiloride-sensitive short-circuit current (Isc) and transepithelial conductance (Gt), whereas no significant effects of PGE2 were observed in hyposmotic serosal solutions. Subsequent addition of mucosal amiloride reduced Isc by approximately 95% and Gt by approximately 60%. Inhibition of endogenous PGE2 production by blockers of phospholipase A2 activity (quinacrine or 3[4-octadecyl]-benzoylacrylic acid; OBBA), or inhibition of cyclooxygenase activity by indomethacin reduced the stimulation of Isc and Gt by hyposmotic solutions. Addition of forskolin (FSK) or 3-Isobutyl-1-methylxanthine (IBMX) also resulted in approximately twofold increases in the amiloride-sensitive Isc and Gt and abolished the effects of subsequent hyposmotic challenge. The effects of forskolin, PGE2, and hyposmotic challenge were diminished by pretreatment with H89, a protein kinase A (PKA) inhibitor. We conclude that osmotic regulation of sodium channel activity interacts with multiple intracellular signaling pathways, specifically the arachidonic acid metabolic pathway and the cAMP/PKA intracellular messenger cascade.

Phosphorylation site-independent downregulation of Na-pump current in A6 epithelia by protein kinase C. Decrease in Na,K-ATPase cell-surface expression.

Phosphorylation site-independent downregulation of Na-pump current in A6 epithelia by protein kinase C. Decrease in Na,K-ATPase cell-surface expression.

Effect of cadmium on active ion transport and cytotoxicity in cultured renal epithelial cells (A6)

A cultured epithelial cell line from toad kidney (A6) was used to study the mechanism by which cadmium (Cd) affects transepithelial resistance (TER) and active transepithelial ion transport measured as short-circuit current (SCC) in vitro. The influence of Cd on cell integrity was investigated by measuring time-dependent TER under controlled conditions and the half-maximal inhibition concentration (IC 50) 24 hr after exposure to 1 m m CdCl 2. The data suggest that Cd deterioration of cell integrity is stronger when applied to the apical relative to the basolateral solution (IC 50 = 173.9 and 147.8 μ M, respectively). Also, the data demonstrate that addition of Cd to the basolateral solution results in a prompt and transient stimulation of the active ion transport from 2.6 ± 0.4 to 8.7 ± 1.1 μA/cm 2. Use of the sodium channel blocker amiloride indicate that Na transport is not involved in Cd-stimulated SCC. Substitution of Cl with SO 4 2− in the basolateral solution and use of the Cl channel inhibitors, diphenylamine-2-carboxylase (DPC) and niflumic acid indicate strongly that Cd increases Cl secretion in A6 epithelium. Thapsigargin (TG), an intracellular Ca-ATPase blocker, inhibits Cd-stimulated active ion transport indicating that Ca-activated Cl channels are probably involved. Therefore, we suggest that Cd by interaction with the basolateral membrane, become internalized and increase Ca intracellularly. In a dose- and time-dependent way an increase in Ca activates specific Cl channels leading to an increased SCC. Thereafter, the increase in Ca leads to disruption of tight junctions thereby decreasing TER. This may lead to deterioration of cell integrity and perhaps even cell death.

Substrate-dependent expression of Na+ transport and shunt conductance in A6 epithelia.

A6 epithelia grown in tissue culture vary enormously in their baseline rates of Na+ transport due to differences in growth media, serum, and other unknown factors. To evaluate the effect(s) of substrates on expression of Na+ transport, we determined short-circuit currents, open-circuit voltages, and electrical resistances of mature confluent A6 epithelia grown on a variety of commercially available permeable supports. Because the cells, growth conditions, and all other factors were the same, differences in transport could be attributed alone to the substrate on which the cells were grown. Tissues were grown on both large- and small-diameter inserts of the same type with differing ratios of edge length to area so that the contribution of the edge and tight junction conductances to the combined shunt conductance of the inserts could be evaluated. Shunt and cellular conductances and the cellular Thévenin electromotive force were determined after aldosterone stimulation and amiloride inhibition of Na+ transport. Marked and extreme differences were observed not only for expression of Na+ transport (controls, 0.09-3.94 microA/cm2; aldosterone, 1.53-28.2 microA/cm2) due to changes of apical membrane conductance but also for the development of junctional conductances (3,250 to < infinity omega.cm2) and edge conductances (13,175 to < infinity omega.cm) among substrates.