Norepinephrine stimulates the epithelial Na+ channel in cortical collecting duct cells via α2-adrenoceptors

There is good evidence for a causal link between excessive sympathetic drive to the kidney and hypertension. We hypothesized that sympathetic regulation of tubular Na+ absorption may occur in the aldosterone-sensitive distal nephron, where the fine tuning of renal Na+ excretion takes place. Here, the appropriate regulation of transepithelial Na+ transport, mediated by the amiloride-sensitive epithelial Na+ channel (ENaC), is critical for blood pressure control. To explore a possible effect of the sympathetic transmitter norepinephrine on ENaC-mediated Na+ transport, we performed short-circuit current ( Isc) measurements on confluent mCCDcl1 murine cortical collecting duct cells. Norepinephrine caused a complex Isc response with a sustained increase of amiloride-sensitive Isc by ∼44%. This effect was concentration dependent and mediated via basolateral α2-adrenoceptors. In cells pretreated with aldosterone, the stimulatory effect of norepinephrine was reduced. Finally, we demonstrated that noradrenergic nerve fibers are present in close proximity to ENaC-expressing cells in murine kidney slices. We conclude that the sustained stimulatory effect of locally elevated norepinephrine on ENaC-mediated Na + absorption may contribute to the hypertensive effect of increased renal sympathetic activity.

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Endothelial-derived nitric oxide inhibits sodium transport by affecting apical membrane channels in cultured collecting duct cells.

Journal of the American Society of Nephrology ◽

10.1681/asn.v4111855 ◽

1994 ◽

Vol 4 (11) ◽

pp. 1855-1860

Author(s):

B A Stoos ◽

O A Carretero ◽

J L Garvin

Keyword(s):

Nitric Oxide ◽

Maximum Rate ◽

Apical Membrane ◽

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Sodium Absorption ◽

Cortical Collecting Duct ◽

Duct Cells ◽

Collecting Duct Cells

Previously, it has been shown that the addition of bradykinin (Bk) to M-1 cortical collecting duct cells in the presence of endothelial cells decreased short-circuit current (Isc), a measure of net active transport. This effect is presumably due to the release of endothelium-derived nitric oxide (EDNO), because the decrease in Isc could be blocked with Nw-nitro-L-arginine. To show that the inhibition of Isc was due to EDNO rather than prostaglandins, the ability of a cyclooxygenase inhibitor to block the inhibition was examined. When Bk was added to cocultures in the presence of meclofenamate (10(-5) M), Isc decreased from 62 +/- 12 to 44.5 +/- 7 muA/cm2, not significantly different from that in the absence of meclofenamate. To determine if the effect was due to an alteration of sodium absorption, Bk (10(-9) M) was added to cocultures, resulting in a decrease in Na flux from 28 +/- 3.1 to 20 +/- 2.2 nEq/min (P < 0.05), with Isc decreasing from 25 +/- 2.4 to 20 +/- 3.6 nEq/min (P < 0.05). To examine if the inhibition was due to blockade at the apical membrane sodium channel or the basolateral Na+/K+ ATPase, the cation-selective ionophore nystatin was used. Nystatin reversed the effect of EDNO on Isc. The effects of EDNO on Na+/K+ ATPase were also measured directly. Under maximum rate conditions, the Na+/K+ ATPase activity of control and Bk-treated cocultures was 5.2 +/- 0.3 and 6.8 +/- 1.0 nmol/min per square centimeter, respectively (not significantly different).(ABSTRACT TRUNCATED AT 250 WORDS)

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P2Y 2 Receptor-mediated Inhibition of Amiloride-sensitive Short Circuit Current in M-1 Mouse Cortical Collecting Duct Cells

The Journal of Membrane Biology ◽

10.1007/s00232-001-0059-4 ◽

2001 ◽

Vol 183 (2) ◽

pp. 115-124 ◽

Cited By ~ 45

Author(s):

J. Thomas ◽

P. Deetjen ◽

W.H. Ko ◽

C. Jacobi ◽

J. Leipziger

Keyword(s):

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Cortical Collecting Duct ◽

Duct Cells ◽

Collecting Duct Cells

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Whole cell and unitary amiloride-sensitive sodium currents in M-1 mouse cortical collecting duct cells

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.4.c998 ◽

1996 ◽

Vol 270 (4) ◽

pp. C998-C1010 ◽

Cited By ~ 7

Author(s):

M. L. Chalfant ◽

T. G. O'Brien ◽

M. M. Civan

Keyword(s):

Collecting Duct ◽

Cell Permeability ◽

Na Channel ◽

Na Channels ◽

Cortical Collecting Duct ◽

Ionic Selectivity ◽

Whole Cell ◽

Duct Cells ◽

Excised Patches ◽

Collecting Duct Cells

Amiloride-sensitive whole cell currents have been reported in M-1 mouse cortical collecting duct cells (Korbmacher et al., J. Gen. Physiol. 102: 761-793, 1993). We have confirmed that amiloride inhibits the whole cell currents but not necessarily the measured whole cell currents. Anomalous responses were eliminated by removing external Na+ and/or introducing paraepithelial shunts. The amiloride-sensitive whole cell currents displayed Goldman rectification. The ionic selectivity sequence of the amiloride-sensitive conductance was Li+ > Na+ >> K+. Growth of M-1 cells on permeable supports increased the amiloride-sensitive whole cell permeability, compared with cells grown on plastic. Single amiloride-sensitive channels were observed, which conformed to the highly selective low-conductance amiloride-sensitive class [Na(5)] of epithelial Na+ channels. Hypotonic pretreatment markedly slowed run-down of channel activity. The gating of the M-1 Na+ channel in excised patches was complex. Open- and closed-state dwell-time distributions from patches that display one operative channel were best described with two or more exponential terms each. We conclude that 1) study of M-1 whole cell Na+ currents is facilitated by reducing the transepithelial potential to zero, 2) these M-1 currents reflect the operation of Na(5) channels, and 3) the Na+ channels display complex kinetics, involving > or = 2 open and > or = 2 closed states.

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Soluble (pro)renin receptor regulation of ENaC involved in aldosterone signaling in cultured collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.00436.2019 ◽

2020 ◽

Vol 318 (3) ◽

pp. F817-F825 ◽

Cited By ~ 4

Author(s):

Fei Wang ◽

Renfei Luo ◽

Kexin Peng ◽

Xiyang Liu ◽

Chuanming Xu ◽

...

Keyword(s):

Transcriptional Activation ◽

Collecting Duct ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Α Subunit ◽

Duct Cells ◽

Nadph Oxidase 1 ◽

Collecting Duct Cells ◽

Two Phases

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na+ channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), the cleavage product of PRR in ENaC regulation, and further tested its relevance to aldosterone signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM within minutes induced a significant and transient increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique. The acute ENaC activation was blocked by the NADPH oxidase 1/4 inhibitor GKT137892 and siRNA against Nox4 but not the β-catenin inhibitor ICG-001. In primary rat inner medullary collecting duct cells, administration of sPRR-His at 10 nM for 24 h induced protein expression of the α-subunit but not β- or γ-subunits of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The transcriptional activation of α-ENaC was dependent on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Ussing chamber determination of short-circuit current showed that aldosterone-induced transepithelial Na+ transport was inhibited by the PRR decoy inhibitor PRO20 and PF-429242, an inhibitor of sPRR-generating enzyme site-1 protease, and the response was restored by the addition of sPRR-His. Medium sPRR was elevated by aldosterone and inhibited by PF-429242. Taken together, these results demonstrate that sPRR induces two phases of ENaC activation via distinct mechanisms and functions as a mediator of the natriferic action of aldosterone.

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Aldosterone regulation of sodium channel gamma-subunit mRNA in cortical collecting duct cells

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.1.c423 ◽

1996 ◽

Vol 271 (1) ◽

pp. C423-C428 ◽

Cited By ~ 33

Author(s):

D. L. Denault ◽

G. Fejes-Toth ◽

A. Naray-Fejes-Toth

Keyword(s):

Nested Pcr ◽

Collecting Duct ◽

Na Channel ◽

Mrna Levels ◽

Cortical Collecting Duct ◽

Gamma Subunit ◽

Subunit Mrna ◽

Duct Cells ◽

Pcr Product ◽

Collecting Duct Cells

Specific regulatory mechanisms of aldosterone-stimulated Na+ reabsorption through the apical amiloride-sensitive channel are unknown. In this study, we examined the effects of aldosterone on Na+ channel gamma-subunit mRNA levels in cultured rabbit cortical collecting duct cells. With the use of reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from aldosterone-treated cells and degenerate primers, a 446-base pair (bp) PCR product was amplified and further characterized by nested PCR and sequencing. The nested PCR yielded a predicted 164-bp product. Sequencing of the 446-bp PCR product revealed 83% nucleotide and 91% amino acid identity to the rat colonic Na+ channel gamma-subunit. The relative abundance of Na+ channel mRNA was determined by quantitative PCR after a 24-h aldosterone treatment. The results demonstrate that Na+ channel gamma-subunit mRNA levels were significantly higher (2.6 +/- 0.42) in aldosterone-treated cultures vs. the controls. This increase, however, is less than the aldosterone-induced increase (3.2 +/- 2.0) in the amiloride-sensitive short-circuit current. These results indicate that Na+ channel gamma-subunit mRNA levels are increased by aldosterone and that this increase is likely to be responsible, at least in part, for the aldosterone-induced Na+ current in the kidney.

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Differential effects of extracellular ATP on chloride transport in cortical collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.00062.2012 ◽

2012 ◽

Vol 303 (4) ◽

pp. F483-F491 ◽

Cited By ~ 12

Author(s):

Madhumitha Rajagopal ◽

Paru P. Kathpalia ◽

Jonathan H. Widdicombe ◽

Alan C. Pao

Keyword(s):

Chloride Transport ◽

Purinergic Signaling ◽

Collecting Duct ◽

Short Circuit ◽

Extracellular Fluid ◽

Extracellular Atp ◽

Transient Increase ◽

Cortical Collecting Duct ◽

Duct Cells ◽

Collecting Duct Cells

Extracellular ATP in the cortical collecting duct can inhibit epithelial sodium channels (ENaC) but also stimulate calcium-activated chloride channels (CACC). The relationship between ATP-mediated regulation of ENaC and CACC activity in cortical collecting duct cells has not been clearly defined. We used the mpkCCDc14 cortical collecting duct cell line to determine effects of ATP on sodium (Na+) and chloride (Cl−) transport with an Ussing chamber system. ATP, at a concentration of 10−6 M or less, did not inhibit ENaC-mediated short-circuit current ( Isc) but instead stimulated a transient increase in Isc. The macroscopic current-voltage relationship for ATP-inducible current demonstrated that the direction of this ATP response changes from positive to negative when transepithelial voltage ( Vte) is clamped to less than −10 mV. We hypothesized that this negative Vte might be found under conditions of aldosterone stimulation. We next stimulated mpkCCDc14 cells with aldosterone (10−6 M) and then clamped the Vte to −50 mV, the Vte of aldosterone-stimulated cells under open-circuit conditions. ATP (10−6 M) induced a transient increase in negative clamp current, which could be inhibited by flufenamic acid (CACC inhibitor) and BAPTA-AM (calcium chelator), suggesting that ATP stimulates Cl− absorption through CACC. Together, our findings suggest that the status of ENaC activity, by controlling Vte, may dictate the direction of ATP-stimulated Cl− transport. This interplay between aldosterone and purinergic signaling pathways may be relevant for regulating NaCl transport in cortical collecting duct cells under different states of extracellular fluid volume.

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ROS production as a common mechanism of ENaC regulation by EGF, insulin, and IGF-1

AJP Cell Physiology ◽

10.1152/ajpcell.00231.2012 ◽

2013 ◽

Vol 304 (1) ◽

pp. C102-C111 ◽

Cited By ~ 37

Author(s):

Daria V. Ilatovskaya ◽

Tengis S. Pavlov ◽

Vladislav Levchenko ◽

Alexander Staruschenko

Keyword(s):

Growth Factor ◽

Nadph Oxidase ◽

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Fine Tuning ◽

Common Mechanism ◽

Ros Production ◽

Cortical Collecting Duct ◽

Nadph Oxidase Complex

The epithelial Na+ channel (ENaC) is a key transporter participating in the fine tuning of Na+ reabsorption in the nephron. ENaC activity is acutely upregulated by epidermal growth factor (EGF), insulin, and insulin-like growth factor-1 (IGF-1). It was also proposed that reactive oxygen species (ROS) have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin, and IGF-1 correlate with ROS production in the mouse cortical collecting duct (mpkCCDc14) cells. Western blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Treatment of mpkCCDc14 cells with EGF, insulin, or IGF-1 evoked an increase in ROS production as measured by CM-H2DCF-DA fluorescence. ROS production caused by a xanthine-xanthine oxidase reaction also resulted in a significant elevation in short-circuit current through the mpkCCDc14 monolayer. Transepithelial current measurements showed an acute increase of amiloride-sensitive current through the mpkCCDc14 monolayer in response to EGF, insulin, or IGF-1. Pretreatment with the nonselective NADPH oxidase activity inhibitor apocynin blunted both ROS production and increase in ENaC-mediated current in response to these drugs. To further test whether NADPH oxidase subunits are involved in the effect of EGF, we used a stable M-1 cell line with a knockdown of Rac1, which is one of the key subunits of the NADPH oxidase complex, and measured amiloride-sensitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdown cells. We hypothesize that EGF, insulin, and IGF-1 have a common stimulatory effect on ENaC mediated by ROS production.

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Regulation of epithelial Na+ channels from M-1 cortical collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.1996.271.4.f861 ◽

1996 ◽

Vol 271 (4) ◽

pp. F861-F870 ◽

Cited By ~ 8

Author(s):

M. L. Chalfant ◽

K. Peterson-Yantorno ◽

T. G. O'Brien ◽

M. M. Civan

Keyword(s):

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Na Channel ◽

Cortical Collecting Duct ◽

Experimental Conditions ◽

Open Time ◽

Epithelial Na Channels ◽

Selective Channel ◽

A Site

The M-1 cell line is derived from the mouse cortical collecting duct and displays the low-conductance, highly Na(+)-selective channel activity of the alpha,beta, gamma-heterotrimeric epithelial Na+ channel (ENaC). The short-circuit current (Isc) across M-1 monolayers was 89 +/- 4 microA/cm2, and the transepithelial conductance was 2.1 +/- 0.2 mS/cm2. Isc was abolished by blocking the Na+ pump with ouabain. Both Isc and transepithelial conductance (gT) were inhibited by benzamil > amiloride >> dimethylamiloride. Under our experimental conditions, vasopressin, vasopressin, forskolin, and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) had no detectable effects on Isc or gT. Increasing apical Na+ entry with nystatin increased Isc. The possible regulation of the M-1 Na+ channel by cAMP-activated protein kinase A (PKA) was further examined with excised inside-out patches. The open-time probability (Po) was not fixed, displaying substantial variance. Perfusion with ATP itself, with the catalytic subunit of PKA with ATP, or with alkaline phosphatase had no consistent effect on Po, the unitary current, or the kinetics of the M-1 Na+ channel. The data are consistent with the concept that PKA stimulates ENaCs by phosphorylating a site with access to but not within the apical membrane patch during cell-attached and excised-patch studies.

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Epidermal growth factor inhibits amiloride-sensitive sodium absorption in renal collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.00028.2002 ◽

2003 ◽

Vol 284 (1) ◽

pp. F57-F64 ◽

Cited By ~ 47

Author(s):

Jie-Pan Shen ◽

Calvin U. Cotton

Keyword(s):

Growth Factor ◽

Chronic Exposure ◽

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Mrna Levels ◽

Erk Pathway ◽

Duct Cells ◽

Collecting Duct Cells ◽

Renal Collecting Duct

The effects of the ERK pathway on electrogenic transepithelial Na+ absorption by renal collecting duct cells were determined. Approximately 90% of the unstimulated short-circuit current (15 ± 1 μA/cm2, n = 10) across conditionally immortalized murine collecting duct epithelial cells (mCT1) is amiloride sensitive and is likely mediated by apical epithelial Na+ channels. Chronic exposure (24 h) of the epithelial monolayers to either EGF (50 ng/ml) or transforming growth factor-α (TGF-α; 20 ng/ml) reduced amiloride-sensitive short-circuit current by >60%. The inhibitory effect of EGF on Na+ absorption was not due to inhibition of basolateral Na+-K+-ATPase, because the pump current elicited by permeabilization of apical membrane with nystatin was not reduced by EGF. Chronic exposure of the mCT1 cells to EGF (20 ng/ml, 24 h) elicited a 70–85% decrease in epithelial Na+ channel subunit mRNA levels. Exposure of mCT1 cells to either EGF (20 ng/ml) or PMA (150 nM) induced rapid phosphorylation of p42/p44 (ERK1/2) and pretreatment of the monolayers with PD-98059 (an ERK kinase inhibitor; 30 μM) prevented phosphorylation of p42/p44. Similarly, pretreatment of mCT1 monolayers with PD-98059 prevented the EGF- and PMA-induced inhibition of amiloride-sensitive Na+ absorption. The results of these studies demonstrate that amiloride-sensitive Na+ absorption by renal collecting duct cells is regulated by the ERK pathway. This pathway may play a role in alterations in ion transport that occur in polycystic kidney disease.

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