Limits...
cAMP increases density of ENaC subunits in the apical membrane of MDCK cells in direct proportion to amiloride-sensitive Na(+) transport.

Morris RG, Schafer JA - J. Gen. Physiol. (2002)

Bottom Line: The density of ENaC subunits was quantified by specific binding of (125)I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4-8 nM.Stimulation with cAMP (20 microM 8-p-chlorothio-cAMP plus 200 microM IBMX) significantly increased AS-I(sc) from 11.2 +/- 1.3 to 18.1 +/- 1.3 microA/cm(2).We interpret the proportional increases in binding and AS-I(sc) to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the I(sc) increase produced by cAMP.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Antidiuretic hormone and/or cAMP increase Na(+) transport in the rat renal collecting duct and similar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was undertaken to determine if that increment in Na(+) transport could be explained quantitatively by an increased density of ENaC Na(+) channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retrovirally transfected with rat alpha-, beta-, and gammaENaC subunits, each of which were labeled with the FLAG epitope in their extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and B.C. Rossier. 1996. PROC: Natl. Acad. Sci. USA. 93:15370-15375). The density of ENaC subunits was quantified by specific binding of (125)I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4-8 nM. Transepithelial Na(+) transport was measured as the amiloride-sensitive short-circuit current (AS-I(sc)) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS-I(sc) measured in the same experiments. Stimulation with cAMP (20 microM 8-p-chlorothio-cAMP plus 200 microM IBMX) significantly increased AS-I(sc) from 11.2 +/- 1.3 to 18.1 +/- 1.3 microA/cm(2). M2 binding (at 1.7 nM M2) increased in direct proportion to AS-I(sc) from 0.62 +/- 0.13 to 1.16 +/- 0.18 fmol/cm(2). Based on the concentration dependence of M2 binding, the quantity of Na(+) channels per unit of AS-I(sc) was calculated to be the same in the presence and absence of cAMP, 0.23 +/- 0.04 and 0.21 +/-0.05 fmol/microA, respectively. These values would be consistent with a single channel conductance of approximately 5 pS (typically reported for ENaC channels) only if the open probability is <0.02, i.e., less than one-tenth of the typical value. We interpret the proportional increases in binding and AS-I(sc) to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the I(sc) increase produced by cAMP.

Show MeSH

Related in: MedlinePlus

Effect of Cl− on the response of short-circuit current (Isc) to cAMP stimulation. (A) Time course of Isc response to cAMP treatment in DMEM and chloride-free medium. Isc was measured across monolayers of αFβFγF MDCK cells induced overnight with 2 mM butyrate and 1 μM dexamethasone. A mixture of 20 μM CPT-cAMP and 200 μM IBMX was added at the time indicated, followed by 20 μM amiloride. (B) Average Isc in experiments described in A. Six experiments were conducted in DMEM and 20 in chloride-free medium. Basal Isc was measured just before, and the cAMP Isc just after the addition of 20 μM CPT-cAMP plus 200 μM IBMX, at the point of maximal response, 5–10 min in DMEM and 10–20 min in chloride-free medium. The final measurement was made 1–3 min after the addition of 20 μM amiloride.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2311399&req=5

fig5: Effect of Cl− on the response of short-circuit current (Isc) to cAMP stimulation. (A) Time course of Isc response to cAMP treatment in DMEM and chloride-free medium. Isc was measured across monolayers of αFβFγF MDCK cells induced overnight with 2 mM butyrate and 1 μM dexamethasone. A mixture of 20 μM CPT-cAMP and 200 μM IBMX was added at the time indicated, followed by 20 μM amiloride. (B) Average Isc in experiments described in A. Six experiments were conducted in DMEM and 20 in chloride-free medium. Basal Isc was measured just before, and the cAMP Isc just after the addition of 20 μM CPT-cAMP plus 200 μM IBMX, at the point of maximal response, 5–10 min in DMEM and 10–20 min in chloride-free medium. The final measurement was made 1–3 min after the addition of 20 μM amiloride.

Mentions: The effect of cAMP on transepithelial transport in αFβFγF MDCK cells was examined in experiments measuring Isc in Ussing chambers. Fig. 5 A compares the time course Isc of representative experiments with αFβFγF MDCK cells in DMEM and chloride-free media, and the average results of several experiments are presented in Fig. 5 B. In DMEM, basal Isc was significantly stimulated from 8.1 ± 1.0 to a peak value of 18.9 ± 2.2 μA/cm2 by the addition of 20 μM 8-CPT-cAMP plus 200 μM IBMX to the apical and basolateral media. There was rapid spike in Isc followed by a broader peak, which decayed over 30 min. Addition of 20 μM amiloride to the apical solution rapidly reduced Isc to ∼5 μA/cm2. The effect of amiloride was statistically significant and confirmed the presence of amiloride-sensitive, cAMP-stimulated Na+ transport in αFβFγF MDCK cells. As also shown in Fig. 5 A, in chloride-free medium the time course of 20 μM 8-CPT-cAMP plus 200 μM IBMX action was quite different. Isc rose from 7.4 ± 1.0 to 13.6 ± 1.2 μA/cm2 without the initial spike observed in DMEM. The cAMP-stimulated current also decayed more slowly in chloride-free medium. Again, addition of 20 μM amiloride to the apical side significantly reduced Isc to ∼3 μA/cm2.


cAMP increases density of ENaC subunits in the apical membrane of MDCK cells in direct proportion to amiloride-sensitive Na(+) transport.

Morris RG, Schafer JA - J. Gen. Physiol. (2002)

Effect of Cl− on the response of short-circuit current (Isc) to cAMP stimulation. (A) Time course of Isc response to cAMP treatment in DMEM and chloride-free medium. Isc was measured across monolayers of αFβFγF MDCK cells induced overnight with 2 mM butyrate and 1 μM dexamethasone. A mixture of 20 μM CPT-cAMP and 200 μM IBMX was added at the time indicated, followed by 20 μM amiloride. (B) Average Isc in experiments described in A. Six experiments were conducted in DMEM and 20 in chloride-free medium. Basal Isc was measured just before, and the cAMP Isc just after the addition of 20 μM CPT-cAMP plus 200 μM IBMX, at the point of maximal response, 5–10 min in DMEM and 10–20 min in chloride-free medium. The final measurement was made 1–3 min after the addition of 20 μM amiloride.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2311399&req=5

fig5: Effect of Cl− on the response of short-circuit current (Isc) to cAMP stimulation. (A) Time course of Isc response to cAMP treatment in DMEM and chloride-free medium. Isc was measured across monolayers of αFβFγF MDCK cells induced overnight with 2 mM butyrate and 1 μM dexamethasone. A mixture of 20 μM CPT-cAMP and 200 μM IBMX was added at the time indicated, followed by 20 μM amiloride. (B) Average Isc in experiments described in A. Six experiments were conducted in DMEM and 20 in chloride-free medium. Basal Isc was measured just before, and the cAMP Isc just after the addition of 20 μM CPT-cAMP plus 200 μM IBMX, at the point of maximal response, 5–10 min in DMEM and 10–20 min in chloride-free medium. The final measurement was made 1–3 min after the addition of 20 μM amiloride.
Mentions: The effect of cAMP on transepithelial transport in αFβFγF MDCK cells was examined in experiments measuring Isc in Ussing chambers. Fig. 5 A compares the time course Isc of representative experiments with αFβFγF MDCK cells in DMEM and chloride-free media, and the average results of several experiments are presented in Fig. 5 B. In DMEM, basal Isc was significantly stimulated from 8.1 ± 1.0 to a peak value of 18.9 ± 2.2 μA/cm2 by the addition of 20 μM 8-CPT-cAMP plus 200 μM IBMX to the apical and basolateral media. There was rapid spike in Isc followed by a broader peak, which decayed over 30 min. Addition of 20 μM amiloride to the apical solution rapidly reduced Isc to ∼5 μA/cm2. The effect of amiloride was statistically significant and confirmed the presence of amiloride-sensitive, cAMP-stimulated Na+ transport in αFβFγF MDCK cells. As also shown in Fig. 5 A, in chloride-free medium the time course of 20 μM 8-CPT-cAMP plus 200 μM IBMX action was quite different. Isc rose from 7.4 ± 1.0 to 13.6 ± 1.2 μA/cm2 without the initial spike observed in DMEM. The cAMP-stimulated current also decayed more slowly in chloride-free medium. Again, addition of 20 μM amiloride to the apical side significantly reduced Isc to ∼3 μA/cm2.

Bottom Line: The density of ENaC subunits was quantified by specific binding of (125)I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4-8 nM.Stimulation with cAMP (20 microM 8-p-chlorothio-cAMP plus 200 microM IBMX) significantly increased AS-I(sc) from 11.2 +/- 1.3 to 18.1 +/- 1.3 microA/cm(2).We interpret the proportional increases in binding and AS-I(sc) to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the I(sc) increase produced by cAMP.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Antidiuretic hormone and/or cAMP increase Na(+) transport in the rat renal collecting duct and similar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was undertaken to determine if that increment in Na(+) transport could be explained quantitatively by an increased density of ENaC Na(+) channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retrovirally transfected with rat alpha-, beta-, and gammaENaC subunits, each of which were labeled with the FLAG epitope in their extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and B.C. Rossier. 1996. PROC: Natl. Acad. Sci. USA. 93:15370-15375). The density of ENaC subunits was quantified by specific binding of (125)I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4-8 nM. Transepithelial Na(+) transport was measured as the amiloride-sensitive short-circuit current (AS-I(sc)) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS-I(sc) measured in the same experiments. Stimulation with cAMP (20 microM 8-p-chlorothio-cAMP plus 200 microM IBMX) significantly increased AS-I(sc) from 11.2 +/- 1.3 to 18.1 +/- 1.3 microA/cm(2). M2 binding (at 1.7 nM M2) increased in direct proportion to AS-I(sc) from 0.62 +/- 0.13 to 1.16 +/- 0.18 fmol/cm(2). Based on the concentration dependence of M2 binding, the quantity of Na(+) channels per unit of AS-I(sc) was calculated to be the same in the presence and absence of cAMP, 0.23 +/- 0.04 and 0.21 +/-0.05 fmol/microA, respectively. These values would be consistent with a single channel conductance of approximately 5 pS (typically reported for ENaC channels) only if the open probability is <0.02, i.e., less than one-tenth of the typical value. We interpret the proportional increases in binding and AS-I(sc) to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the I(sc) increase produced by cAMP.

Show MeSH
Related in: MedlinePlus