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.

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)

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.

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.

K+ transport in the mesonephric collecting duct system of the toadBufo bufo

2002 ◽  
Vol 205 (7) ◽  
pp. 897-904
Author(s):  
Nadja Møbjerg ◽  
Erik Hviid Larsen ◽  
Ivana Novak
Keyword(s):  
Cell Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
K Channel ◽  
Duct System ◽  
Apical Cell Membrane ◽  
Collecting Ducts ◽  
Collecting Tubules ◽  
K Transport

SUMMARYWe studied the mechanisms of K+ transport in cells from isolated and perfused collecting tubules and ducts from the mesonephric kidney of the toad Bufo bufo. Cells were impaled with microelectrodes across the basal cell membrane. The basolateral membrane potential(Vbl) depolarized upon change of bath [K+] from 3 to 20 mmol l-1 demonstrating a large K+ conductance in this membrane. In collecting tubules and collecting ducts a Vbl of -66±2 mV and -74±4 mV depolarized by 30±2 mV and 36±3 mV, respectively (N=23; 15). The K+ channel inhibitor Ba2+ (1 mmol l-1)inhibited the basolateral K+ conductance and depolarized a Vbl of -64±4 mV by 30±6 mV (N=8). Luminal K+ steps (3 to 20 mmol l-1) demonstrated a K+ conductance in the apical cell membrane. In collecting tubules and collecting ducts a Vbl of -70±3 mV and-73±3 mV depolarized by 11±3 mV and 16±3 mV, respectively(N=11; 11). This conductance could also be inhibited by Ba2+, which depolarized a Vbl of -71±5 mV by 9±3 mV (N=5). The pump inhibitor ouabain (1 mmol l-1) depolarized Vbl, but addition of furosemide to bath solution did not affect Vbl. The[K+] in urine varied from 1.3 to 22.8 mmol l-1. In conclusion, we propose that the collecting duct system of B. bufosecretes K+ into the urine via luminal K+channels.

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+.

The collecting tubule of Amphiuma. I. Electrophysiological characterization

1987 ◽  
Vol 253 (6) ◽  
pp. F1263-F1272 ◽  
Author(s):  
M. Hunter ◽  
J. D. Horisberger ◽  
B. Stanton ◽  
G. Giebisch
Keyword(s):  
Cell Membrane ◽  
Cell Model ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Potassium Conductance ◽  
Sodium Reabsorption ◽  
Apical Cell Membrane ◽  
Cation Selectivity ◽  
Basolateral Cell Membrane ◽  
Collecting Tubules

Single collecting tubules of Amphiuma kidneys were perfused in vitro to characterize their electrophysiological properties. The lumen-negative potential (-24 mV) was abolished by amiloride in the lumen and by ouabain in the bath. Ion substitution experiments in the lumen demonstrated the presence of a large sodium conductance in the apical cell membrane, but no evidence was obtained for a significant potassium or chloride conductance. Ion substitutions in the bath solution and the depolarizing effect of barium on the basolateral membrane potential demonstrated the presence of a large potassium conductance in the basolateral cell membrane. Measurements of dilution potentials in amiloride-treated tubules revealed a modest cation selectivity of the paracellular pathway. These results support a cell model in which sodium reabsorption occurs by electrodiffusion across the apical cell membrane and active transport across the basolateral cell membrane. The absence of a detectable potassium conductance in the apical cell membrane suggests that secretion of this ion cannot take place by diffusion from cell to lumen.

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

1986 ◽  
Vol 250 (1) ◽  
pp. F70-F76 ◽  
Author(s):  
B. M. Koeppen
Keyword(s):  
Cell Membrane ◽  
Collecting Duct ◽  
Apical Cell ◽  
Rabbit Kidney ◽  
Cell Type ◽  
Apical Cell Membrane ◽  
Basolateral Cell Membrane ◽  
Outer Stripe ◽  
Medullary Collecting Duct ◽  
Conductive Properties

Segments of the outer medullary collecting duct were dissected from the outer stripe of the rabbit kidney (OMCDo) 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. The transepithelial voltage (VT) and resistance (RT) averaged -10.7 +/- 2.5 mV, lumen negative, and 28.5 +/- 2.9 k omega X cm (n = 27), respectively. Two cell types could be defined by their electrophysiological properties. One cell type (n = 7) had a mean basolateral membrane voltage (Vbl) of -30.1 +/- 2.4 mV, a fractional resistance of the apical membrane (fRa = Ra/Ra + Rbl) near unity (0.99 +/- 0.01), and a predominantly Cl(-)-selective basolateral cell membrane. The second cell type (n = 27) had a mean Vbl of -63.7 +/- 2.7 mV, a fRa of 0.81 +/- 0.02, and a predominantly K+-selective basolateral cell membrane. The present study focused on defining the conductive properties of this latter cell type. Amiloride (10(-5) M) and BaCl2 (2 mM) were used as probes of apical cell membrane Na+ and K+ conductive pathways, respectively. Amiloride increased fRa from 0.80 +/- 0.02 to 0.98 +/- 0.01 (n = 12), whereas BaCl2 increased fRa from 0.77 +/- 0.03 to 0.82 +/- 0.03 (n = 9). The conductive properties of the basolateral cell membrane were assessed by ion substitutions of the bath solution. A 10-fold increase in the bath [K+] depolarized Vbl by 34.9 +/- 1.9 mV (n = 16) in less than 1 s, indicating that this membrane was predominantly K+ selective.(ABSTRACT TRUNCATED AT 250 WORDS)

H-K-ATPase activity in PNA-binding intercalated cells of newborn rabbit cortical collecting duct

1997 ◽  
Vol 272 (2) ◽  
pp. F167-F177 ◽  
Author(s):  
A. Constantinescu ◽  
R. B. Silver ◽  
L. M. Satlin
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Adult Rabbit ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Load ◽  
Ethanesulfonic Acid

Functional and immunocytochemical studies indicate that intercalated cells in the adult rabbit cortical collecting duct (CCD) possess an H-K-adenosinetriphosphatase (H-K-ATPase). Because growing subjects must retain K+ and excrete H+, we sought to determine whether H-K-ATPase is present in the CCD early in life and, if so, to assess its activity and polarity. H-K-ATPase activity was defined as the initial rate of Sch-28080-inhibitable K+-dependent cell pH (pHi) recovery observed, in the absence of Na+, in response to an in vitro acid load. Transporter activity was assayed in intercalated cells labeled with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and apical cell surface marker rhodamine peanut lectin (PNA) in split-open CCDs isolated from neonatal and adult New Zealand White rabbits. In Na+-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions (nominal absence of CO2/HCO3-), the rate of K+-dependent pH(i) recovery from a NH4Cl-induced acid load was similar in newborn (0.056 +/- 0.015 pH U/min, n = 9) and adult (0.060 +/- 0.019 pH U/min; n = 9, P = not significant) cells. This rate of K+-dependent pH(i) recovery was significantly reduced by 10-20 pM Sch-28080, an inhibitor of gastric H-K-ATPase, in both newborns (0.009 +/- 0.003 pH U/min, n = 7) and adults (0.013 +/- 0.007 pH U/min, n = 9) (P < 0.05 compared with rates in absence of inhibitor). To determine whether the location of the transporter is consistent with a role in K+ absorption and H+ secretion, pH(i) recovery of acutely acid-loaded intercalated cells in neonatal CCDs (n = 7) microperfused and bathed in the absence of Na+ and K+ was monitored after selective addition of K+ to either the luminal or basolateral membrane. Addition of 5 mM K+ led to a significantly greater rate of pH(i) recovery when it was added to the luminal rather than the peritubular solution (0.049 +/- 0.005 vs. 0.018 +/- 0.005 pH U/min, P < 0.05). We conclude that PNA-binding intercalated cells of the neonatal CCD possess H-K-ATPase activity, predominantly located in the apical membrane. This provides a mechanism for H secretion and K+ retention, processes required for growth.

Stimulation of total CO2 flux by 10% CO2 in rabbit CCD: role of an apical Sch-28080- and Ba-sensitive mechanism

1994 ◽  
Vol 267 (1) ◽  
pp. F114-F120 ◽  
Author(s):  
X. Zhou ◽  
C. S. Wingo
Keyword(s):  
Collecting Duct ◽  
Co2 Flux ◽  
K Channel ◽  
Co2 Absorption ◽  
Cortical Collecting Duct ◽  
Atpase Inhibitor ◽  
Contrast Exposure ◽  
Stimulation Of

These studies examine the effect of ambient PCO2 on net bicarbonate (total CO2) absorption by the in vitro perfused cortical collecting duct (CCD) from K-replete rabbits and the mechanism responsible for this effect. Exposure to 10% CO2 increased net bicarbonate flux (total CO2 flux, JtCO2) by 1.8-fold (P < 0.005), and this effect was inhibited by luminal 10 microM Sch-28080, an H-K-adenosinetriphosphatase (H-K-ATPase) inhibitor. In contrast, exposure to 10% CO2 significantly decreased Rb efflux, and this decrement in Rb efflux was blocked by luminal 2 mM Ba, a K channel blocker. Thus transepithelial tracer Rb flux did not increase upon exposure to 10% CO2 as we have observed in this segment under K-restricted conditions. The observation that 10% CO2 increased net bicarbonate absorption without a change in absorptive Rb flux suggested that 10% CO2 increased apical K recycling. To test this hypothesis, we examined whether luminal Ba inhibited the stimulation of luminal acidification induced by 10% CO2. If apical K exit were necessary for full activation of proton secretion, then inhibiting K exit should indirectly affect the stimulation of JtCO2 by 10% CO2. In fact, the effect of 10% CO2 on JtCO2 in the presence of 2 mM luminal Ba was quantitatively indistinguishable from the effect of 10% CO2 on JtCO2 in the presence of 10 microM luminal Sch-28080.(ABSTRACT TRUNCATED AT 250 WORDS)

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