scholarly journals Depletion of Cholesterol Reduces ENaC Activity by Decreasing Phosphatidylinositol-4,5-Bisphosphate in Microvilli

2018 ◽  
Vol 47 (3) ◽  
pp. 1051-1059 ◽  
Author(s):  
Yu-Jia Zhai ◽  
Bing-Chen Liu ◽  
Shi-Peng Wei ◽  
Chu-Fang Chou ◽  
Ming-Ming Wu ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Single Channel ◽  
Collecting Duct ◽  
Critical Role ◽  
Apical Cell ◽  
Underlying Mechanism ◽  
Systemic Blood Pressure ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Apical Cell Membrane

Background/Aims: The epithelial sodium channel (ENaC) in cortical collecting duct (CCD) principal cells plays a critical role in regulating systemic blood pressure. We have previously shown that cholesterol (Cho) in the apical cell membrane regulates ENaC; however, the underlying mechanism remains unclear. Methods: Patch-clamp technique and confocal microscopy were used to evaluate ENaC activity and density. Results: Here we show that extraction of membrane Cho with methyl-β-cyclodextrin (MβCD) significantly reduced amiloride-sensitive current and ENaC single-channel activity. The effects were reproduced by inhibition of Cho synthesis in the cells with lovastatin. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP2), an ENaC activator, is predominantly located in the microvilli, a specialized apical membrane domain. Here, our confocal microscopy data show that α-ENaC was co-localized with PIP2 in the microvilli and that Cho was also co-localized with PIP2 in the microvilli. Either extraction of Cho with MβCD or inhibition of Cho synthesis with lovastatin consistently reduced the levels of Cho, PIP2, and ENaC in the microvilli. Conclusions: Since PIP2 can directly stimulate ENaC and also affect ENaC trafficking, these data suggest that depletion of Cho reduces ENaC apical density and activity at least in part by decreasing PIP2 in the microvilli.

Mineralocorticoid regulation of apical cell membrane Na+ and K+ transport of the cortical collecting duct

1985 ◽  
Vol 248 (6) ◽  
pp. F858-F868 ◽  
Author(s):  
S. C. Sansom ◽  
R. G. O'Neil
Keyword(s):  
Cell Membrane ◽  
Tight Junction ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Apical Cell ◽  
Cortical Collecting Duct ◽  
Apical Cell Membrane ◽  
K Secretion ◽  
Junction Conductance ◽  
K Transport

The effects of mineralocorticoid (DOCA) treatment of rabbits on the Na+ and K+ transport properties of the cortical collecting duct apical cell membrane were assessed using microelectrode techniques. Applying standard cable techniques and equivalent circuit analysis to the isolated perfused tubule, the apical cell membrane K+ and Na+ currents and conductances could be estimated from the selective effects of the K+ channel blocker Ba2+ and the Na+ channel blocker amiloride on the apical membrane; amiloride treatment was observed also to decrease the tight junction conductance by an average of 10%. After 1 day of DOCA treatment, the Na+ conductance and current (Na+ influx) of the apical cell membrane doubled and remained elevated with prolonged treatment for up to 2 wk. The apical cell membrane K+ conductance was not influenced after 1 day, although the K+ current (K+ secretion) increased significantly due to an increased driving force for K+ exit. After 4 days or more of DOCA treatment the K+ conductance doubled, resulting in a further modest stimulation in K+ secretion. After 2 wk of DOCA treatment the tight junction conductance decreased by near 30%, resulting in an additional hyperpolarization of the transepithelial voltage, thereby favoring K+ secretion. It is concluded that the acute effect (within 1 day) of mineralocorticoids on Na+ and K+ transport is an increase in the apical membrane Na+ conductance followed by delayed chronic alterations in the apical membrane K+ conductance and tight junction conductance, thereby resulting in a sustained increased capacity of the tubule to reabsorb Na+ and secrete K+.

Critical role of the mineralocorticoid receptor in aldosterone-dependent and aldosterone-independent regulation of ENaC in the distal nephron

2021 ◽  
Author(s):  
Viatcheslav Nesterov ◽  
Marko Bertog ◽  
Jérémie Canonica ◽  
Edith Hummler ◽  
Richard Coleman ◽  
...  
Keyword(s):  
Mineralocorticoid Receptor ◽  
Collecting Duct ◽  
Critical Role ◽  
Sodium Absorption ◽  
Cortical Collecting Duct ◽  
Distal Nephron ◽  
Patch Clamp Technique ◽  
Transition Zones ◽  
Rate Limiting Step

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for sodium absorption in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the collecting duct. Previously, we demonstrated that ENaC activity in the DCT2/CNT transition zone is constitutively high and independent of aldosterone, in contrast to its aldosterone dependence in the late CNT and initial cortical collecting duct (CNT/CCD). The mineralocorticoid receptor (MR) is expressed in the entire ASDN. Its activation by glucocorticoids is prevented through 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) abundantly expressed in the late but probably not the early part of ASDN. We hypothesized that ENaC function in the early part of the ASDN is aldosterone-independent but may depend on MR activated by glucocorticoids due to low 11β-HSD2 abundance. To test this hypothesis, we used doxycycline-inducible nephron-specific MR-deficient mice (MR KO). Whole-cell ENaC currents were investigated in isolated nephron fragments from DCT2/CNT or CNT/CCD transition zones using the patch-clamp technique. ENaC activity was detectable in CNT/CCD of control mice but absent or barely detectable in the majority of CNT/CCD preparations from MR KO mice. Importantly, ENaC currents in DCT2/CNT were greatly reduced in MR KO mice compared to ENaC currents in DCT2/CNT of control mice. Immunofluorescence for 11β-HSD2 was abundant in CCD, less prominent in CNT and very low in DCT2. We conclude that MR is critically important for maintaining aldosterone-independent ENaC activity in DCT2/CNT. Aldosterone-independent MR activation is probably mediated by glucocorticoids due to low expression of 11β-HSD2.

Microelectrode assessment of chloride-conductive properties of cortical collecting duct

1984 ◽  
Vol 247 (2) ◽  
pp. F291-F302 ◽  
Author(s):  
S. C. Sansom ◽  
E. J. Weinman ◽  
R. G. O'Neil
Keyword(s):  
Cell Membrane ◽  
Tight Junction ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Membrane Conductance ◽  
Cortical Collecting Duct ◽  
Apical Cell Membrane ◽  
Conductive Properties ◽  
Chloride Activity

The chloride-conductive properties of the isolated rabbit cortical collecting duct were assessed with microelectrode techniques. The transepithelial, apical, and basolateral membrane potential differences, Vte, Va, and Vb, respectively, were monitored continuously along with periodic measurements of the transepithelial conductance, Gte, and fractional resistance, fRa (ratio of apical to apical plus basolateral membrane resistance). Active transport was eliminated in all experiments by luminal addition of 50 microM amiloride in HCO3-free solutions. Upon reducing the chloride activity in the bath (gluconate replacement), there was a marked depolarization of Vb and decrease in Gte and fRa, demonstrating a major dependence of the basolateral membrane conductance on the bath chloride activity. However, a significant K+ conductance at that barrier was also apparent since raising the bath K+ concentration caused an increase in Gte and fRa and depolarization of Vb. Lowering the chloride activity of the perfusate caused a consistent decrease of Gte but not of fRa, effects consistent with a high C1- conductance of the tight junction and little, if any, apical membrane C1- conductance. By use of the C1- -dependent conductances, the C1- permeabilities at equilibrium were estimated to be near 1.0 X 10(-5) cm X s-1 for the tight junction, PtiC1, and 5 X 10(-5) cm X s-1 for the basolateral cell membrane, PbC1. It is concluded that the paracellular pathway provides a major route for transepithelial C1- transport. Furthermore, since the isotopically measured C1- permeability is severalfold greater than PtiC1, a significant transcellular flux of C1- must exist, implicating a neutral exchange mechanism at the apical cell membrane in series with the high basolateral membrane C1- conductance.

Ca2+-activated K+ channels in cultured medullary thick ascending limb cells

1987 ◽  
Vol 252 (2) ◽  
pp. C121-C127 ◽  
Author(s):  
S. E. Guggino ◽  
W. B. Guggino ◽  
N. Green ◽  
B. Sacktor
Keyword(s):  
Cell Membrane ◽  
Single Channel ◽  
Apical Cell ◽  
Outward Current ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Patch Clamp Technique ◽  
Apical Cell Membrane ◽  
K Channels ◽  
Medullary Thick Ascending Limb

The conductive properties of a clone of medullary thick ascending limb (MTAL) cells (GRB-MAL1) were assessed using conventional microelectrodes and the patch clamp technique. The apical cell membrane potential (Va) of MTAL cells was -46 +/- 3 mV. Addition of Ba2+ (1 mM) to the apical solution induced a 22 +/- 2 mV depolarization of Va, whereas furosemide hyperpolarized Va by -5 +/- 1 mV. In the cell-attached patch configuration, the most frequently occurring channel had a single channel conductance of 121 +/- 5 pS and carried outward current. In excised patches, current movement was down the electrochemical K+ gradient. Fluctuations were activated by depolarization of Va and by increasing Ca2+ concentration on the intracellular face. Micromolar amounts of Ba2+ on the intracellular face of the membrane inhibited channel activity. We conclude that cultures of MTAL cells GRB-MAL1 retain at least two of the properties of the mature phenotype, namely, an apical K+ conductance and a sensitivity to loop diuretics; the most frequently occurring channel in the apical cell membrane is a Ca2+-activated, maxi-K+ channel; and, finally Ca2+-activated K+ channels may play a role in generating the apical K+ conductance in cultured MTAL cells.

Characterization of apical cell membrane Na+ and K+ conductances of cortical collecting duct using microelectrode techniques

1984 ◽  
Vol 247 (1) ◽  
pp. F14-F24 ◽  
Author(s):  
R. G. O'Neil ◽  
S. C. Sansom
Keyword(s):  
Cell Membrane ◽  
Collecting Duct ◽  
Apical Cell ◽  
Membrane Voltage ◽  
Cortical Collecting Duct ◽  
Apical Cell Membrane ◽  
Basolateral Cell Membrane ◽  
Luminal Side ◽  
Conductive Properties ◽  
Microelectrode Techniques

The apical cell membrane ionic conductive properties of the isolated perfused rabbit cortical collecting duct (tubule) were assessed at 37 degrees C using microelectrode techniques. In the initial evaluation of the methodology, it was observed that stable cell membrane voltage recordings could be obtained by impaling cells either from the luminal side across the apical cell membrane or from the bath side across the basolateral cell membrane, providing initial evidence supporting the application of these techniques to this tissue. With the latter method of impalement, it was observed that addition of amiloride (50 microM) to the luminal perfusate caused a hyperpolarization of the apical cell membrane voltage, a decrease in the transepithelial conductance, and an increase in the fractional resistance (estimated as the ratio of the resistance of the apical cell membrane to the sum of apical and basolateral cell membrane resistances). These results are consistent with an amiloride-sensitive Na+ conductance at the apical cell border. In a similar manner it was deduced from the effects of elevating K+ in the luminal perfusate from 5 to either 25 or 50 mM that there was a high K+ conductance at the apical border. This conductive pathway was blocked by the luminal addition of 5 mM Ba2+ or reduction of the luminal pH to 4.0. Furthermore, since addition of both amiloride and Ba2+ to the perfusate caused the fractional resistance to increase from 0.52 +/- 0.04 to 0.91 +/- 0.03, the Na+ and K+ conductances are the apparent dominant conductive pathways at that border. It is concluded that microelectrode techniques can be applied successfully to the cortical collecting duct and that the apical cell membrane possesses an amiloride-sensitive Na+ conductance and a Ba2+- and H+-sensitive K+ conductance.

Intracellular microelectrode characterization of the rabbit cortical collecting duct

1983 ◽  
Vol 244 (1) ◽  
pp. F35-F47 ◽  
Author(s):  
B. M. Koeppen ◽  
B. A. Biagi ◽  
G. H. Giebisch
Keyword(s):  
Cell Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Fold Increase ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Apical Cell Membrane ◽  
Basolateral Cell Membrane

Cortical collecting ducts of the rabbit were perfused in vitro and the intracellular potential (Vbl) was measured with KCl-filled microelectrodes. The ratio of apical to basolateral membrane resistance (Ra/Rbl) was estimated from the voltage divider ratio using cable analysis. In control tubules Vbl averaged--84.0 +/- 2.5 mV and Ra/Rbl was 0.83 +/- 0.11. Pretreatment of the rabbits with mineralocorticoid caused Vbl to hyperpolarize to--105.8 +/- 3.1 mV and Ra/Rbl to decrease slightly to 0.62 +/- 0.10. A 10-fold increase of the luminal [K+] caused a 40.6 +/- 3.1 mV depolarization of Vbl in control tubules and a 33.0 +/- 4.2 mV depolarization in tubules from DOCA-pretreated rabbits. Concurrently, Ra/Rbl decreased in both groups, consistent with the existence of a conductive K+ channel at the apical cell membrane. This apical K+ channel was not sensitive to amiloride but was blocked by Ba2+. Conductive movement of Na+ across the apical membrane was also apparent in that Ra/Rbl increased with amiloride from 0.61 +/- 0.10 to 1.45 +/- 0.28. A 10-fold increase in the bath [K+] caused a 28.6 +/- 3.8 and a 49.4 +/- 4.4 mV depolarization of Vbl in tubules obtained from control and DOCA-pretreated rabbits, respectively. In both groups Ra/Rbl increased, suggesting that the basolateral cell membrane also contains a conductive K+ channel. Taken together the results support a model in which the transepithelial reabsorption of Na+ and the transepithelial secretion of K+ are driven by the Na+-K+-ATPase located in the basolateral cell membrane, with passive movement of these ions occurring through separate conductive pathways in the apical cell membrane.

Involvement of actin cytoskeleton in modulation of apical K channel activity in rat collecting duct

1994 ◽  
Vol 267 (4) ◽  
pp. F592-F598 ◽  
Author(s):  
W. H. Wang ◽  
A. Cassola ◽  
G. Giebisch
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Collecting Duct ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Channel Inhibition ◽  
Inside Out

We have employed the patch-clamp technique to investigate the role of the actin cytoskeleton in the modulation of the low-conductance K+ channel in the apical membrane of the rat cortical collecting duct (CCD). This K+ channel is inactivated by application of cytochalasin B or D, both compounds known to disrupt actin filaments. The effect of both cytochalasins, B and D, was fully reversible in cell-attached patches, but channel activity could not be fully restored in excised membrane patches. The effect of cytochalasins on channel activity was specific and resulted from depolymerization of the actin cytoskeleton, since application of 10 microM chaetoglobosin C, a cytochalasin analogue that does not depolymerize the actin filaments, had no effect on channel activity in inside-out patches. Addition of either actin monomers or of the polymerizing actin filaments in inside-out patches to the cytosolic medium had no effect on channel activity. This suggests that cytochalasin B- or D-induced inactivation of apical K+ channels is not caused by obstruction of the channel pore by actin. We also observed that channel inhibition by cytochalasin B or D could be blocked by pretreatment with 5 microM phalloidin, a compound that stabilizes actin filaments. We conclude that apical K+ channel activity depends critically on the integrity of the actin cytoskeleton.

Conductive properties of a rabbit cortical collecting duct cell line: regulation by isoproterenol

1992 ◽  
Vol 262 (4) ◽  
pp. F578-F582 ◽  
Author(s):  
P. Dietl ◽  
N. Kizer ◽  
B. A. Stanton
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cell Line ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Conductive Properties ◽  
Cl Conductance ◽  
Kinase A

The present study was carried out to characterize the membrane conductive properties of RCCT-28A cells, a continuous cell line derived from rabbit cortical collecting duct (CCD). RCCT-28A cells have many phenotypic properties of acid-secreting intercalated cells (A-IC). Using the whole cell patch-clamp technique, we found that the cells are conductive to Cl-, but not to Na+ or K+. The beta-adrenergic agonists isoproterenol (2 x 10(-6) M) and adenosine 3',5'-cyclic monophosphate (cAMP, 10(-4) M) increased the whole cell Cl- conductance. Protein kinase A (150 nM) in the patch pipette (i.e., intracellular solution) also increased whole cell Cl- conductance. Because isoproterenol increases cAMP levels in these cells, we conclude that isoproterenol stimulates the Cl- conductance by increasing cell cAMP, which in turn activates protein kinase A. In contrast, vasopressin does not increase cAMP in these cells and did not increase the Cl- conductance. In conclusion, these experiments show that RCCT-28A cells, like A-IC, are conductive only to Cl-. Thus RCCT-28A cells are a good model with which to study Cl- channels in the collecting duct.

Conductive properties of the rabbit outer medullary collecting duct: inner stripe

1985 ◽  
Vol 248 (4) ◽  
pp. F500-F506 ◽  
Author(s):  
B. M. Koeppen
Keyword(s):  
Cell Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Rabbit Kidney ◽  
Apical Cell Membrane ◽  
Basolateral Cell Membrane ◽  
Medullary Collecting Duct ◽  
Conductive Properties

Segments of outer medullary collecting duct were dissected from the inner stripe of the rabbit kidney (OMCDi) and perfused in vitro. The conductive properties of the tubule epithelium and individual cell membranes were determined by means of cable analysis and intracellular voltage-recording microelectrodes. In 35 tubules the transepithelial voltage (VT) and resistance (RT) averaged 17.2 +/- 1.4 mV, lumen positive, and 58.6 +/- 5.3 k omega X cm, respectively. The basolateral membrane voltage, (Vbl) was -29.2 +/- 2.1 mV (n = 23). The apical cell membrane did not contain appreciable ion conductances, as evidenced by the high values of apical cell membrane fractional resistance (fRa = Ra/Ra + Rb), which approached unity (0.99 +/- 0.01; n = 23). Moreover, addition of amiloride or BaCl2 to the tubule lumen was without effect on the electrical characteristics of the cell, as was a twofold reduction in luminal [Cl-]. The conductive properties of the basolateral cell membrane were assessed with bath ion substitutions. A twofold reduction in bath [Cl-] depolarized Vbl by 14.7 +/- 0.4 mV (theoretical, 17 mV), while a 10-fold increase in bath [K+] resulted in only a 0.9 +/- 0.4 mV depolarization (theoretical, 61 mV). Substituting bath Na+ with tetramethylammonium (from 150 to 75 mM) was without effect. Reducing bath [HCO-3] from 25 to 5 mM (constant PCO2) resulted in a steady-state depolarization of Vbl of 8.4 +/- 0.4 mV that could not be attributed to conductive HCO-3 movement. Thus, the basolateral cell membrane is predominantly Cl- selective.(ABSTRACT TRUNCATED AT 250 WORDS)

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