scholarly journals Mechanisms of increased Na+ transport in ATII cells by cAMP: we agree to disagree and do more experiments

2000 ◽  
Vol 278 (2) ◽  
pp. L233-L238 ◽  
Author(s):  
Ahmed Lazrak ◽  
Vance G. Nielsen ◽  
Sadis Matalon
Keyword(s):  
Short Circuit ◽  
Alveolar Epithelium ◽  
Alveolar Type ◽  
Single Channels ◽  
Open Probability ◽  
Apical Membranes ◽  
Camp Levels ◽  
Inside Out ◽  
Cytoplasmic Side ◽  
Short Circuit Currents

Existing evidence supports the presence of active transport of Na+ across the mammalian alveolar epithelium and its upregulation by agents that increase cytoplasmic cAMP levels. However, there is controversy regarding the mechanisms responsible for this upregulation. Herein we present the results of various patch-clamp studies indicating the presence of 25- to 27-pS, amiloride-sensitive, moderately selective Na+ channels (Na+-to-K+ permeability ratio = 7:1) located on the apical membranes of rat alveolar type II (ATII) cells maintained in primary culture. The addition of terbutaline to the bath solution increased the open probability of single channels present in cell-attached patches of ATII cells without affecting their conductance. A similar increase in open probability was seen after the addition of protein kinase A, ATP, and Mg2+ to the cytoplasmic side of inside-out patches. Measurement of short-circuit currents across confluent monolayers of rat or rabbit ATII cells indicates that terbutaline and 8-(4-chlorophenylthio)-cAMP increase vectorial Na+ transport and activate Cl− channels. Currently, there is a controversy as to whether the cAMP-induced increase in Na+ transport is due solely to hyperpolarization of the cytoplasmic side of the ATII cell membrane due to Cl− influx or whether it results from simultaneous stimulation of both Cl− and Na+ conductive pathways. Additional studies are needed to resolve this issue.

Role of SGK1 in nitric oxide inhibition of ENaC in Na+-transporting epithelia

2005 ◽  
Vol 289 (3) ◽  
pp. C717-C726 ◽  
Author(s):  
My N. Helms ◽  
Ling Yu ◽  
Bela Malik ◽  
Dean J. Kleinhenz ◽  
C. Michael Hart ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Epithelial Cells ◽  
Single Channel ◽  
Short Circuit ◽  
Primary Cultures ◽  
Alveolar Epithelium ◽  
Alveolar Type ◽  
No Production ◽  
Open Probability ◽  
No Donor

Several studies have shown that nitric oxide (NO) inhibits Na+ transport in renal and alveolar monolayers. However, the mechanisms by which NO alters epithelial Na+ channel (ENaC) activity is unclear. Therefore, we examined the effect of applying the NO donor drug l-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) to cultured renal epithelial cells. A6 and M1 cells were maintained on permeable supports in medium containing 1.5 μM dexamethasone and 10% bovine serum. After 1.5 μM PAPA-NONOate was applied, amiloride-sensitive short-circuit current measurements decreased 29% in A6 cells and 44% in M1 cells. This differed significantly from the 3% and 19% decreases in A6 and M1 cells, respectively, treated with control donor compound ( P < 0.0005). Subsequent application of PAPA-NONOate to amiloride-treated control (no NONOate) A6 and M1 cells did not further decrease transepithelial current. In single-channel patch-clamp studies, NONOate significantly decreased ENaC open probability ( Po) from 0.186 ± 0.043 to 0.045 ± 0.009 ( n = 7; P < 0.05) without changing the unitary current. We also showed that aldosterone significantly decreased NO production in primary cultures of alveolar type II (ATII) epithelial cells. Because inducible nitric oxide synthase (iNOS) coimmunoprecipitated with the serum- and glucocorticoid-inducible kinase (SGK1) and both proteins colocalized in the cytoplasm (as shown in our studies in mouse ATII cells), SGK1 may also be important in regulating NO production in the alveolar epithelium. Our study also identified iNOS as a novel SGK1 phosphorylated protein (at S733 and S903 residues in miNOS) suggesting that one way in which SGK1 could increase Na+ transport is by altering iNOS production of NO.

scholarly journals Mechanisms of Regulation of Epithelial Sodium Channel by SGK1 in A6 Cells

2004 ◽  
Vol 124 (4) ◽  
pp. 395-407 ◽  
Author(s):  
Diego Alvarez de la Rosa ◽  
Teodor G. Păunescu ◽  
Willem J. Els ◽  
Sandy I. Helman ◽  
Cecilia M. Canessa
Keyword(s):  
Sodium Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Epithelial Sodium Channel ◽  
Sodium Reabsorption ◽  
Epithelial Cell Line ◽  
Open Probability ◽  
Renal Epithelial Cell ◽  
Intracellular Compartments ◽  
Apical Membranes

The serum and glucocorticoid induced kinase 1 (SGK1) participates in the regulation of sodium reabsorption in the distal segment of the renal tubule, where it may modify the function of the epithelial sodium channel (ENaC). The molecular mechanism underlying SGK1 regulation of ENaC in renal epithelial cells remains controversial. We have addressed this issue in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible system. Expression of a constitutively active mutant of SGK1 (SGK1TS425D) induced a sixfold increase in amiloride-sensitive short-circuit current (Isc). Using noise analysis we demonstrate that SGK1 effect on Isc is due to a fourfold increase in the number of functional ENaCs in the membrane and a 43% increase in channel open probability. Impedance analysis indicated that SGK1TS425D increased the absolute value of cell equivalent capacitance by an average of 13.7%. SGK1TS425D also produced a 1.6–1.9-fold increase in total and plasma membrane subunit abundance, without changing the half-life of channels in the membrane. We conclude that in contrast to aldosterone, where stimulation of transport can be explained simply by an increase in channel synthesis, SGK1 effects are more complex and involve at least three actions: (1) increase of ENaC open probability; (2) increase of subunit abundance within apical membranes and intracellular compartments; and (3) activation of one or more pools of preexistent channels within the apical membranes and/or intracellular compartments.

Modulation of bioelectric properties across alveolar type II cells by substratum

1989 ◽  
Vol 257 (4) ◽  
pp. C678-C688 ◽  
Author(s):  
G. R. Cott
Keyword(s):  
Cell Morphology ◽  
Short Circuit ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Capacitance Effects ◽  
Short Circuit Currents ◽  
Cells Cultured

Rat alveolar type II cells were cultured on collagen-coated filters (CCF) and human amnionic basement membrane (ABM) to determine the effect of culture substratum on the development of monolayer bioelectric properties. Monolayers cultured on both substrata rapidly developed bioelectric properties with similar time courses, monolayer capacitance values (approximately 1 muF/cm2), current-voltage relationships, and responses to stimulants and inhibitors of active ion transport. Increasing seeding densities tended to increase monolayer bioelectric properties regardless of culture substratum. Monolayers cultured on ABM had higher resistance values (491 vs. 291 omega.cm2) and lower short-circuit currents (2.85 vs. 4.51 muA/cm2) than monolayers with similar cell densities cultured on CCF. These differences in monolayer bioelectric properties were not due to differences in substratum resistance or capacitance effects. The relationships between monolayer bioelectric properties were also affected by the culture substratum. In additional experiments, cells cultured on contracted gels formed monolayers with high short-circuit currents (9.25 muA/cm2). Cell morphology varied depending on the culture substratum, with cells cultured on contracted gels appearing the most cuboidal, whereas the flattest and most attenuated cells were those cultured on ABM. On the basis of these observations, we conclude that culture substratum significantly affects the development of bioelectric properties across alveolar type II cell monolayers. In vivo the bioelectric properties across the alveolar epithelium may also vary with changes in cellular substratum or cell density (e.g., after acute lung injury) and possibly with cell morphology (e.g., alveolar type I vs. alveolar type II cells).

scholarly journals Light-dependent K+ Channels in the Mollusc Onchidium Simple Photoreceptors Are Opened by cGMP

2002 ◽  
Vol 120 (4) ◽  
pp. 581-597 ◽  
Author(s):  
Tsukasa Gotow ◽  
Takako Nishi
Keyword(s):  
K Channel ◽  
Single Channels ◽  
Open Probability ◽  
Mean Values ◽  
K Channels ◽  
Open Time ◽  
Hyperpolarizing Response ◽  
The Mean ◽  
K Concentration ◽  
Inside Out

Light-dependent K+ channels underlying a hyperpolarizing response of one extraocular (simple) photoreceptor, Ip-2 cell, in the marine mollusc Onchidium ganglion were examined using cell-attached and inside-out patch-clamp techniques. A previous report (Gotow, T., T. Nishi, and H. Kijima. 1994. Brain Res. 662:268–272) showed that a depolarizing response of the other simple photoreceptor, A-P-1 cell, results from closing of the light-dependent K+ channels that are activated by cGMP. In the cell-attached patch recordings of Ip-2 cells, external artificial seawater (ASW) was replaced with a modified ASW containing 150 mM K+ and 200 mM Mg2+ to suppress any synaptic input and to maintain the membrane potential constant. When Ip-2 cells were equilibrated with this modified ASW, the internal K+ concentration was estimated to be 260 mM. Light-dependent single-channels in the cell-attached patch on these cells were opened by light but scarcely by voltage. After confirming the light-dependent channel activity in the cell-attached patches, an application of cGMP to the excised inside-out patches newly activated a channel that disappeared on removal of cGMP. Open and closed time distributions of this cGMP-activated channel could be described by the sum of two exponents with time constants τo1, τo2 and τc1, τc2, respectively, similar to those of the light-dependent channel. In both the channels, τo1 and τo2 in ms ranges were similar to each other, although τc2 over tens of millisecond ranges was different. τo1, τo2, and the mean open time τo were both independent of light intensity, cGMP concentration, and voltage. In both channels, the open probability increased as the membrane was depolarized, without changing any of τo2 or τo. In both, the reversal potentials using 200- and 450-mM K+-filled pipettes were close to the K+ equilibrium potentials, suggesting that both the channels are primarily K+ selective. Both the mean values of the channel conductance were estimated to be the same at 62 and 91 pS in 200- and 450-mM K+ pipettes at nearly 0 mV, respectively. Combining these findings with those in the above former report, it is concluded that cGMP is a second messenger which opens the light-dependent K+ channel of Ip-2 to cause hyperpolarization, and that the channel is the same as that of A-P-1 closed by light.

β-Adrenergic regulation of amiloride-sensitive lung sodium channels

2002 ◽  
Vol 282 (4) ◽  
pp. L609-L620 ◽  
Author(s):  
Xi-Juan Chen ◽  
Douglas C. Eaton ◽  
Lucky Jain
Keyword(s):  
Intracellular Calcium ◽  
Sodium Transport ◽  
Lung Epithelial Cells ◽  
Alveolar Type ◽  
Intracellular Camp ◽  
Adrenergic Stimulation ◽  
Open Probability ◽  
Calcium Sequestration ◽  
Selective Channel ◽  
Camp Levels

We investigated the mechanism by which cAMP increases sodium transport in lung epithelial cells. Alveolar type II (ATII) cells have two types of amiloride-sensitive, cation channels: a nonselective cation channel (NSC) and a highly selective channel (HSC). Exposure of ATII cells to cAMP, β-adrenergic agonists, or other agents that increase adenylyl cyclase activity increased activity of both channel types, albeit by different mechanisms. NSC open probability ( P o) increased severalfold when exposed to terbutaline, isoproterenol, forskolin, or cAMP analogs without any change in NSC number. In contrast, terbutaline increased HSC number with no significant change in HSC P o. For both channels, the effect of terbutaline was blocked by propranolol and H-89, suggesting a protein kinase A (PKA) requirement for β-adrenergic-induced changes in channel activity. Terbutaline increased cAMP levels in ATII cells, but intracellular calcium also increased. Calcium sequestration with BAPTA blocked β-adrenergic-induced increases in NSC P o but did not alter HSC activity. These observations suggest that β-adrenergic stimulation increases intracellular cAMP and activates PKA. PKA increases HSC number and increases intracellular calcium. The increase in calcium increases NSC P o. Thus increased cAMP levels are likely to increase lung sodium transport regardless of which channel type is present.

cAMP activation of chloride and fluid secretion across the rabbit alveolar epithelium

1998 ◽  
Vol 275 (6) ◽  
pp. L1127-L1133 ◽  
Author(s):  
Vance G. Nielsen ◽  
Michael D. Duvall ◽  
Manuel S. Baird ◽  
Sadis Matalon
Keyword(s):  
Short Circuit ◽  
Alveolar Epithelium ◽  
Short Circuit Current ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Intracellular Camp ◽  
Type Ii ◽  
Alveolar Fluid Clearance ◽  
Ussing Chambers

Active Na+ transport by alveolar epithelial cells has been demonstrated to contribute significantly to alveolar fluid clearance. However, the contribution of transepithelial Cl− movement to the reabsorption of isosmotic fluid across the alveolar epithelium in vivo has not been elucidated. We hypothesized that Cl− transport could be increased across the alveolar epithelium in vivo and across cultured alveolar type II cells by agents that increase intracellular cAMP (e.g., forskolin). In studies where 5% albumin in sodium methanesulfonate (a Cl−-free solution) was administered into the lung, forskolin administration significantly increased intracellular influx of Cl− and fluid into the alveolar space. In vitro studies with cultured rabbit alveolar type II cell monolayers in Ussing chambers demonstrated that elevations in intracellular cAMP increase short-circuit current by increasing both Cl−secretion and Na+ reabsorption. The cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide and the loop diuretic bumetanide partially decreased the forskolin-induced increase in short-circuit current. These data may explain the failure of agonist that stimulated intracellular cAMP to increase alveolar fluid clearance in the rabbit. Moreover, the data suggest that in the event Na+absorptive pathways are damaged, transepithelial Cl− secretion and the consequent intra-alveolar fluid influx may be upregulated.

Nitric oxide inhibits Na+absorption across cultured alveolar type II monolayers

1998 ◽  
Vol 274 (3) ◽  
pp. L369-L377 ◽  
Author(s):  
Yi Guo ◽  
Michael D. Duvall ◽  
John P. Crow ◽  
Sadis Matalon
Keyword(s):  
Nitric Oxide ◽  
Inhibitory Concentration ◽  
Short Circuit ◽  
Alveolar Type ◽  
Type Ii ◽  
Cyclic Monophosphate ◽  
Apical Side ◽  
Apical Membranes ◽  
Spermine Nonoate ◽  
Permeable Supports

We examined the mechanisms by which nitric oxide (⋅ NO) decreased vectorial Na+ transport across confluent monolayers of rat alveolar type II (ATII) cells grown on permeable supports. Amiloride (10 μM) applied to the apical side of monolayers inhibited ∼90% of the equivalent ( I eq) and the short-circuit ( I sc) current, with an half-maximal inhibitory concentration (IC50) of 0.85 μM, indicating that Na+ entry into ATII cells occurred through amiloride-sensitive Na+ channels. ⋅ NO generated by spermine NONOate and papa NONOate added to both sides of the monolayers decreased I eq and increased transepithelial resistance in a concentration-dependent fashion (IC50 = 0.4 μM ⋅ NO). These changes were prevented or reversed by addition of oxyhemoglobin (50 μM). Incubation of ATII monolayers with 8-bromoguanosine 3′,5′-cyclic monophosphate (400 μM) had no effect on transepithelial Na+ transport. When the basolateral membranes of ATII cells were permeabilized with amphotericin B (10 μM) in the presence of a mucosal-to-serosal Na+ gradient (145:25 mM), ⋅ NO (generated by 100 μM papa NONOate) inhibited ∼60% of the amiloride-sensitive I sc. In addition, after permeabilization of the apical membranes, ⋅ NO inhibited the I sc[a measure of Na+-K+-adenosinetriphosphatase (ATPase) activity] by ∼60%. We concluded that ⋅ NO at noncytotoxic concentrations decreased Na+ absorption across cultured ATII monolayers by inhibiting both the amiloride-sensitive Na+ channels and Na+-K+-ATPase through guanosine 3′,5′-cyclic monophosphate-independent mechanisms.

scholarly journals Membrane cholesterol extraction decreases Na+ transport in A6 renal epithelia

2006 ◽  
Vol 290 (1) ◽  
pp. C87-C94 ◽  
Author(s):  
Corina Balut ◽  
Paul Steels ◽  
Mihai Radu ◽  
Marcel Ameloot ◽  
Willy Van Driessche ◽  
...  
Keyword(s):  
Steady State ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Cholesterol Content ◽  
Membrane Cholesterol ◽  
Open Probability ◽  
Renal Epithelia ◽  
Pump Activity ◽  
Apical Membranes

In this study, we have investigated the dependence of Na+ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current ( Isc), transepithelial conductance ( GT), and transepithelial capacitance ( CT) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl-β-cyclodextrin (mβCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mβCD treatment. However, apical mβCD application hampered the responses of Isc and GT to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in Isc demonstrated that apical mβCD treatment decreased the epithelial Na+ channel (ENaC) open probability ( Po) in the steady state as well as after activation of Na+ transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by CT measurements. Na+ transport activation by hypotonicity was abolished during basolateral mβCD treatment as a result of reduced Na+/K+ pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na+/K+ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na+ transport by reducing ENaC Po.

scholarly journals Effects of insulin and phosphatase on a Ca2(+)-dependent Cl- channel in a distal nephron cell line (A6).

1990 ◽  
Vol 95 (5) ◽  
pp. 773-789 ◽  
Author(s):  
Y Marunaka ◽  
D C Eaton
Keyword(s):  
Cell Line ◽  
Chloride Channels ◽  
Single Channel ◽  
Single Channels ◽  
Distal Nephron ◽  
Open Probability ◽  
Inward Current ◽  
Cl Channel ◽  
Relationship Of ◽  
Inside Out

A Cl- channel with a small single-channel conductance (3 pS) was observed in cell-attached patches formed on the apical membrane of cells from the distal nephron cell line (A6) cultured on permeable supports. The current-voltage (I-V) relationship from cell-attached patches or inside-out patches with 1 microM cytosolic Ca2+ strongly rectified with no inward current at potentials more negative than ECl. However, the rectification decreased (i.e., inward current increased) when the cytosolic Ca2+ concentration ([Ca2+]i) was increased above 1 microM. If [Ca2+]i is increased to 800 microM, the I-V relationship became linear. Besides the change in the I-V relationship, an increase in [Ca2+]i also increases the open probability of the channel. Regardless of the recording condition, the channel has one open and one closed state. Both closing and opening rates were dependent on [Ca2+]i; an increase of [Ca2+]i decreased the closing rate and increased the opening rate. The Ca2+ dependence of transition rates at positive membrane potentials (cell interior with respect to external surface) were much larger than the dependence at negative intracellular potentials. The I-V relationship of chloride channels in inside-out patches from cells pretreated with insulin was linear even with 1 microM [Ca2+]i, while channel currents from cells under similar conditions but without insulin still strongly rectified. Alkaline phosphatase applied to the intracellular surface of inside-out patches altered the outward rectification of single channels in a manner qualitatively similar to that of insulin pretreatment. These observations suggest that phosphorylation/dephosphorylation of the channel modulates the sensitivity of the Cl- channel to cytosolic Ca2+ and that insulin produces its effect by promoting dephosphorylation of the channel.

Sign in / Sign up

Export Citation Format

Share Document