Transepithelial capacitance decrease reveals closure of lateral interspace in A6 epithelia

1999 ◽  
Vol 437 (5) ◽  
pp. 680-690 ◽  
Author(s):  
W. Van Driessche ◽  
Rita De Vos ◽  
Danny Jans ◽  
Jeannine Simaels ◽  
Patrick De Smet ◽  
...  
Keyword(s):  
A6 Epithelia

Use of cultured epithelia to study transport and its regulation

1983 ◽  
Vol 106 (1) ◽  
pp. 55-69
Author(s):  
J. S. Handler
Keyword(s):  
Cell Biology ◽  
Culture Conditions ◽  
Millipore Filter ◽  
A6 Epithelia ◽  
Amphibian Urinary Bladder ◽  
Polarized Epithelia ◽  
Cultured Epithelia ◽  
Camp Levels ◽  
Epithelia Transport ◽  
Standard Techniques

Epithelial cells from a variety of species and organs form polarized epithelia in culture. When epithelia are grown on a porous surface, such as a millipore filter, transport can be studied using adaptations of standard techniques. In the few years in which cultured epithelia have been studied by transport physiologists, most work has been focused on identification and description of the differentiated transport exhibited by cultured epithelia. Epithelia formed by a continuous line of cells derived from pig kidney (LLC-PK1) exhibit sodium-coupled glucose transport similar to that of the proximal tubule and have vasopressin-sensitive adenylate cyclase that has been studied in great detail. Also of interest are epithelia formed by continuous lines of cells derived from amphibian kidney (A6) and from amphibian urinary bladder (TBM). Each line forms epithelia that have high electrical resistance and amiloride-sensitive sodium transport. Transport is stimulated by aldosterone and by cAMP or hormones that raise cell cAMP levels. In LLC-PK1 and in A6 epithelia, transport and the response to hormones can be manipulated by manipulating the culture conditions. Cultured epithelia have also been used to explore the cell biology of epithelia. Most interesting in this regard are studies of the development and maintenance of epithelial cell polarity. This approach should be especially valuable.

Time-dependent apical membrane K+ and Na+ selectivity in cultured kidney cells

1987 ◽  
Vol 253 (1) ◽  
pp. C1-C6 ◽  
Author(s):  
S. R. Thomas ◽  
E. Mintz
Keyword(s):  
Apical Membrane ◽  
Time Dependent ◽  
Kidney Cells ◽  
K Channels ◽  
Membrane Selectivity ◽  
Conductive Pathway ◽  
Group I ◽  
A6 Epithelia ◽  
Group Ii ◽  
Apical Membranes

Intracellular microelectrodes were used to study apical membrane selectivity to Na+ and K+ of cultured toad kidney cells (A6) grown on permeable supports. Membrane selectivity was tested by responses of apical membrane potential to replacement of Na+ by K+ or tetraethylammonium and by addition of amiloride to perfusion solutions. The A6 epithelia fell into two groups: those with K+-selective apical membranes, lack of amiloride sensitivity, and near-zero transepithelial potential (group I); and those with Na+-selective apical membranes and a serosa-positive, amiloride-sensitive transepithelial potential (Vm----s; group II). The transition from group I to group II behavior appeared definitive and time dependent, occurring approximately 10 days after plating onto filters. Transepithelial measurements under sterile conditions showed that overnight incubation with aldosterone (10(-7) M), after development of tight junctions (transepithelial resistance elevated) but before development of significant Vm----s, induced the switch from group I to group II behavior. Apical addition of Ba2+, a known blocker of K+ channels, unexpectedly reduced transepithelial resistance (Rm----s) in group I and group II A6, suggesting that it not only blocked K+ channels (when they are present) but may also open a parallel conductive pathway. In summary, after approximately 10 days in culture, apical membranes of A6 epithelia undergo a switch from K+ to Na+ selectivity, overnight incubation with aldosterone can trigger this change, and finally, Ba2+ may open a paracellular conductive pathway.

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

1997 ◽  
Vol 273 (2) ◽  
pp. C434-C441 ◽  
Author(s):  
S. I. Helman ◽  
X. Liu
Keyword(s):  
Short Circuit ◽  
Small Diameter ◽  
Membrane Conductance ◽  
Growth Conditions ◽  
Edge Length ◽  
Open Circuit ◽  
Growth Media ◽  
Na Transport ◽  
A6 Epithelia ◽  
Open Circuit Voltages

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 Thevenin 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.

Role of PKCα in feedback regulation of Na+transport in an electrically tight epithelium

2002 ◽  
Vol 283 (4) ◽  
pp. C1122-C1132 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Justin D. Platzer ◽  
Roxanne L. Reger ◽  
Abderrahmane Bengrine
Keyword(s):  
Steady State ◽  
Blot Analysis ◽  
Feedback Regulation ◽  
Feedback Mechanism ◽  
Mrna Levels ◽  
Protein Levels ◽  
Pkc Isoforms ◽  
A6 Epithelia ◽  
Tight Epithelia ◽  
Stimulation Of

It has long been known that Na+ channels in electrically tight epithelia are regulated by homeostatic mechanisms that maintain a steady state and allow new levels of transport to be sustained in hormonally challenged cells. Little is known about the potential pathways involved in these processes. In addition to short-term effect, recent evidence also indicates the involvement of PKC in the long-term regulation of the epithelial Na+ channel (ENaC) at the protein level (40). To determine whether stimulation of ENaC involves feedback regulation of PKC levels, we utilized Western blot analysis to determine the distribution of PKC isoforms in polarized A6 epithelia. We found the presence of PKC isoforms in the conventional (α and γ), novel (δ, η, and ε), and atypical (ι, λ, and ζ) groups. Steady-state stimulation of Na+ transport with aldosterone was accompanied by a specific decrease of PKCα protein levels in both the cytoplasmic and membrane fractions. Similarly, overnight treatment with an uncharged amiloride analog (CDPC), a procedure that through feedback regulation causes a stimulation of Na+ transport, also decreased PKCα levels. These effects were additive, indicating separate mechanisms that converge at the level of PKCα. These effects were not accompanied by changes of PKCα mRNA levels as determined by Northern blot analysis. We propose that this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na+ transport.

Adenosine 3',5'-cyclic monophosphate stimulates chloride secretion in A6 epithelia

1986 ◽  
Vol 251 (5) ◽  
pp. C810-C814 ◽  
Author(s):  
M. Yanase ◽  
J. S. Handler
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Apical Surface ◽  
Cyclic Monophosphate ◽  
A6 Epithelia ◽  
In Series ◽  
Cl Channels ◽  
Solution Bathing ◽  
Stimulation Of

Basal and aldosterone-stimulated short-circuit current (Isc) of A6 epithelia are known to be equivalent to net apical to basal Na flux and are completely inhibited by 0.05 mM amiloride added to the solution bathing the apical surface of the epithelium. In the absence of amiloride, the Isc stimulated by adenosine 3',5'-cyclic monophosphate (cAMP) is also equivalent to net apical to basal Na flux. However, amiloride does not completely inhibit the cAMP-stimulated Isc. In this study, the cAMP-stimulated, amiloride-insensitive Isc was characterized, using vasopressin or forskolin to raise cell cAMP. After basal Isc is inhibited by amiloride, forskolin stimulates Isc, conductance, and bidirectional 36Cl flux. Stimulation of Isc depends on the presence of both Na and Cl; stimulation of conductance depends on the presence of Cl. 36Cl flux studies showed that the cAMP-stimulated, amiloride-insensitive Isc is equivalent to net Cl flux. It is inhibited by ouabain and by furosemide or bumetanide added to the solution bathing the basal surface of the epithelium. In view of the effect of cAMP in some other epithelia, we suggest that cAMP activates apical membrane Cl channels that are in series with a Na-K-Cl cotransporter in the basolateral plasma membrane.

Steroid hormone-dependent expression of blockersensitive ENaCs in apical membranes of A6 epithelia

1997 ◽  
Vol 273 (5) ◽  
pp. C1650-C1656 ◽  
Author(s):  
Lynn M. Baxendale-Cox ◽  
Randall L. Duncan ◽  
Xuehong Liu ◽  
Kieron Baldwin ◽  
Willem J. Els ◽  
...  
Keyword(s):  
Growth Medium ◽  
Single Channel ◽  
Apical Membrane ◽  
Short Circuit ◽  
Noise Analysis ◽  
Short Circuit Current ◽  
Growth Media ◽  
Open Probability ◽  
A6 Epithelia ◽  
Single Channel Currents

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 μA/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 ∼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.

Aldosterone action: induction of p21rasandfra-2 and transcription-independent decrease inmyc,jun, andfos

1999 ◽  
Vol 276 (5) ◽  
pp. C1154-C1161 ◽  
Author(s):  
Benjamin Spindler ◽  
François Verrey
Keyword(s):  
Protein Synthesis ◽  
Transcription Factors ◽  
Signaling Cascade ◽  
Mrna Levels ◽  
Gene Products ◽  
Adrenal Steroids ◽  
Ras Protein ◽  
A6 Epithelia ◽  
Receptor Mrnas

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 (∼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.

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

1998 ◽  
Vol 275 (1) ◽  
pp. C189-C199 ◽  
Author(s):  
Patrick De Smet ◽  
Jinqing Li ◽  
Willy Van Driessche
Keyword(s):  
Osmotic Shock ◽  
Regulatory Volume Decrease ◽  
Inhibitory Effect ◽  
Cell Swelling ◽  
Acute Administration ◽  
Noticeable Effect ◽  
Membrane Stretch ◽  
A6 Epithelia ◽  
Volume Recovery

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 with86Rb 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([Formula: see text]) rapidly, reaching a maximum 7 min after the peak in Tc. Quinine (0.5 mM) completely inhibited RVD and [Formula: see text]. 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[Formula: see text]. Acute administration of lanthanides at the time of osmolality decrease did not affect the initial phase of RVD and reduced [Formula: see text]only slightly. Apical Gd3+ exerted an inhibitory effect on RVD and [Formula: see text]. 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[Formula: see text] by ∼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.

scholarly journals Ras Pathway Activates Epithelial Na+ Channel and Decreases Its Surface Expression in Xenopus Oocytes

1998 ◽  
Vol 9 (12) ◽  
pp. 3417-3427 ◽  
Author(s):  
Luca Mastroberardino ◽  
Benjamin Spindler ◽  
Ian Forster ◽  
Jan Loffing ◽  
Roberta Assandri ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Expression System ◽  
Regulatory Protein ◽  
Surface Expression ◽  
Na Channel ◽  
Target Cells ◽  
Ras Pathway ◽  
A6 Epithelia ◽  
The Mean ◽  
Epithelial Na Channel

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 withXK-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-Ras2AG12Vled 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-expressedXENaC was increased byXK-Ras2AG12V. The chemical driving force for Na+ influx was not changed, suggesting thatXK-Ras2AG12V increased the mean activity ofXENaCs 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.

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