NH4Cl inhibition of transport in the rabbit cortical collecting tubule

1985 ◽  
Vol 248 (5) ◽  
pp. F631-F637 ◽  
Author(s):  
L. L. Hamm ◽  
C. Gillespie ◽  
S. Klahr
Keyword(s):  
Cation Transport ◽  
Sodium Reabsorption ◽  
Bathing Solution ◽  
Collecting Tubule ◽  
Potassium Secretion ◽  
Transepithelial Voltage ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

Ammonium has previously been found to inhibit transport in a number of tissues. The present experiments were designed to evaluate the effect of ammonium chloride on transepithelial voltage (VTE) and cation transport in the isolated rabbit cortical collecting tubule perfused in vitro. Peritubular NH4Cl (2-10 mM) inhibited VTE in these tubules independent of bath or lumen pH. Luminal NH4Cl had a similar effect. However, VTE did not change with bath NH4Cl in tubules treated with amiloride or ouabain. Furthermore, when bath PCO2 was lowered simultaneously with the addition of NH4Cl to the bath, little change in VTE occurred, raising the possibility that intracellular pH falls after addition of NH4Cl to the bath. Consistent with the voltage effects, 5 mM NH4Cl in the bathing solution inhibited net sodium reabsorption by 36% and potassium secretion by 44%. Unidirectional lumen-to-bath tracer fluxes were consistent with predominant inhibition of the sodium reabsorptive flux and the potassium secretory flux. These findings may have relevance to metabolic acidosis in vivo because ionic ammonium concentrations attain the levels used in this study.

Effects of isoproterenol on potassium secretion by the cortical collecting tubule

1984 ◽  
Vol 246 (6) ◽  
pp. F804-F810 ◽  
Author(s):  
P. L. Kimmel ◽  
S. Goldfarb
Keyword(s):  
Chloride Transport ◽  
Bathing Solution ◽  
Hormonal Stimulation ◽  
Significant Fall ◽  
Collecting Tubule ◽  
Nephron Segment ◽  
Significant Attenuation ◽  
Potassium Secretion ◽  
Transepithelial Voltage ◽  
Cortical Collecting Tubule

These studies tested the effects of isoproterenol on potassium secretion in the isolated perfused cortical collecting tubule. Isoproterenol, 10(-6) M (n = 6) and 10(-4) M (n = 2), added to bathing solution produced a significant fall in potassium secretion [13.5 +/- 1.2 to 8.0 +/- 0.9 peq X mm-1 X min-1 (P less than 0.01)] and in transepithelial voltage (P less than 0.01) compared with time controls (n = 9). Pretreatment with propranolol abolished this effect (n = 4). Addition of propranolol alone to the bath caused no significant change in potassium secretion (n = 8). 8-[p-Chlorophenylthio]cAMP (10(-4) M, isotonic perfusate) added to the bath produced a significant fall in potassium secretion [11.5 +/- 1.7 to 7.2 +/- 1.3 peq X mm-1 X min-1, n = 7 (P less than 0.01)]. Arginine vasopressin (25 microU/ml), which also stimulates adenylate cyclase activity in this segment, had no significant effect on potassium secretion (n = 10). When chloride was replaced by methyl sulfate in all solutions (n = 6), there was a significant attenuation in the fall in potassium secretion in experiments with 10(-6) M isoproterenol compared with experiments with chloride-containing bath solutions (P less than 0.05). These data suggest that isoproterenol has a specific action of reducing potassium secretion in the cortical collecting tubule either through alternating chloride transport per se or through some other effect dependent on the presence of chloride (e.g., hydrogen ion secretion). Also, this effect is probably mediated by cAMP-dependent events. The lack of effect of vasopressin on potassium secretion suggests that separate cells or cellular pools of cAMP are involved in hormonal stimulation by isoproterenol and vasopressin in this nephron segment.

Stimulation of chloride transport by HCO3-CO2 in rabbit cortical collecting tubule

1986 ◽  
Vol 251 (1) ◽  
pp. F49-F56 ◽  
Author(s):  
K. Tago ◽  
V. L. Schuster ◽  
J. B. Stokes
Keyword(s):  
Chloride Transport ◽  
Acid Base ◽  
Cl Transport ◽  
Collecting Tubule ◽  
Acid Base Status ◽  
Stimulation Of ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

We examined both the role of HCO3-CO2 in Cl transport as well as the effect of in vivo acid-base status on Cl transport by the rabbit cortical collecting tubule. The lumen-to-bath 36Cl tracer flux, expressed as the rate coefficient KCl, was measured in either HEPES-buffered (CO2-free) or HCO3-CO2-containing solutions. Amiloride was added to the perfusate to minimize the transepithelial voltage and thus the electrical driving force for Cl diffusion. Because KCl fell spontaneously with time in HCO3-CO2 solutions in the absence but not the presence of cAMP, we used cAMP throughout to avoid time-dependent changes. Acute in vitro removal of bath HCO3-CO2 reduced KCl. Acetazolamide addition in HEPES-buffered solutions also lowered KCl; KCl could be restored to control values by adding exogenous HCO3-CO2 in the presence of acetazolamide. In vivo acid-base effects on Cl transport were determined by dissecting tubules from either NaHCO3-loaded or NH4Cl-loaded rabbits. Tubules from HCO3-loaded rabbits had higher rates of Cl self exchange. Acute in vitro addition of bath HCO3-CO2 increased KCl and did so to a greater degree in tubules from HCO3-loaded rabbits. Most of this effect of HCO3-CO2 addition on KCl could not be accounted for by Cl-HCO3 exchange; rather, it appeared due to stimulation of Cl self exchange. The data are consistent with 36Cl transport occurring via Cl-HCO3 exchange as well as Cl self exchange. Both processes are acutely stimulated by HCO3 and/or Co2, and both are chronically regulated by in vivo acid-base status.

Acidification of luminal fluid by the rabbit cortical collecting tubule perfused in vitro

1982 ◽  
Vol 242 (5) ◽  
pp. F521-F531 ◽  
Author(s):  
B. M. Koeppen ◽  
S. I. Helman
Keyword(s):  
Disulfonic Acid ◽  
Reverse Polarity ◽  
Perfusion Fluid ◽  
Data Support ◽  
Collecting Tubule ◽  
Transepithelial Voltage ◽  
Ph Electrodes ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

The ability of the rabbit cortical collecting tubule to acidify the luminal fluid was determined with double-barreled antimony pH electrodes. In Na+/K+ Ringer the tubules maintained a transepithelial voltage (VToc) of -45.4 +/- 5.5 mV (bath grounded) and a minimum luminal fluid pH of 5.93 +/- 0.11. Chronic mineralocorticoid pretreatment of the rabbits caused the VToc to become more negative (-78.7 +/- 8.2 mV) and decreased the minimum luminal fluid pH to 5.43 +/- 0.16. In most tubules (control and mineralocorticoid-pretreated) the measured pH was more acidic than could be accounted for by either the lumen-negative VToc or CO2 equilibration of the perfusion fluid. When tubules were perfused and bathed in 0 Na+/0 K+ Ringer they developed a lumen-positive VToc, which was stimulated by mineralocorticoid, was sensitive to the PCO2 of the bathing solutions, but was not dependent on Cl- in either the luminal or bath solutions. Luminal acidification in the absence of Na+ and K+ (pH = 6.05 +/- 0.12) occurred against a lumen-positive VToc of +11.5 +/- 1.9 mV. Addition of 10(-4) M ouabain to the bath of tubules studied in Na+/K+ Ringer caused the VToc to reverse polarity and the luminal fluid pH to increase. In contrast, ouabain had no effect on either the lumen-positive VToc or the minimum luminal fluid pH when added to the bath of tubules in 0 Na+/0 K+ Ringer. Bath addition of 10(-4) M acetazolamide and/or 5 X 10(-4) M 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) caused alkalinization of the luminal fluid in tubules studied in either Na+/K+ or 0 Na+/0 K+ Ringer. In 0 Na+/0 K+ Ringer, acetazolamide and SITS reduced the lumen-positive VToc to near zero. The data support the existence of a distinct acidification mechanism in the rabbit cortical collecting tubule, which is both active and electrogenic.

Potassium secretion by the cortical collecting tubule: effect of C1 gradients and ouabain

1989 ◽  
Vol 256 (2) ◽  
pp. F306-F313 ◽  
Author(s):  
C. S. Wingo
Keyword(s):  
Secretory Process ◽  
Electrochemical Gradient ◽  
Na Transport ◽  
Cl Secretion ◽  
Kcl Cotransport ◽  
Collecting Tubule ◽  
K Secretion ◽  
Potassium Secretion ◽  
Transepithelial Voltage ◽  
Cortical Collecting Tubule

In various epithelia K and Cl transport are molecularly coupled and KCl cotransport is dependent on the Na-K pump. The present study examines 1) the effect of a bath-to-lumen Cl gradient on K secretion during active Na transport and 2) the effect of basolateral ouabain on K secretion and Na absorption in the presence and the absence of a bath-to-lumen Cl gradient. Under symmetrical conditions there was significant K secretion (JK = -24.0 +/- 3.9 pmol.mm-1.min-1) and Cl secretion (JCl = -15.7 +/- 3.7 pmol.mm-1.min-1). Transepithelial voltage (VT) was significantly lumen negative (-25.3 +/- 5.9 mV), and Cl secretion occurred against its electrochemical gradient by a transcellular mechanism. Increasing bath [Cl] did not hyperpolarize VT; in fact there was a tendency for VT to depolarize and K secretion was not stimulated. However, ouabain significantly inhibited active Cl secretion and net Na absorption both in the presence and absence of a bath-to-lumen Cl gradient. Furthermore, ouabain totally inhibited K secretion in the absence of external ion gradients but inhibited K secretion by only 50% in the presence of a bath-to-lumen Cl gradient. This ouabain-insensitive K secretion exhibited a codependence on Cl secretion. Thus K secretion may occur passively, utilizing Cl movement down its electrochemical gradient when active Na transport is inhibited by ouabain. The results are compatible with the presence of a Cl-linked K-secretory process in the rabbit CCT.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct Na+-K+ pump stimulation by K+ in cortical collecting tubules: a mechanism for early renal K+ adaptation

1989 ◽  
Vol 257 (4) ◽  
pp. F595-F601 ◽  
Author(s):  
Y. Fujii ◽  
A. I. Katz
Keyword(s):  
Turnover Rate ◽  
Rapid Rise ◽  
Hormonal Factors ◽  
Collecting Tubule ◽  
Collecting Tubules ◽  
Stimulation Of ◽  
External K ◽  
Cortical Collecting Tubule

To evaluate the mechanism of increased Na+-K+ pump turnover rate that characterizes the early cortical collecting tubule (CCT) response to K+ loading [Y. Fujii, S. K. Mujais, and A. I. Katz. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25): F279-F284, 1989.], we measured ouabain-sensitive 86Rb+ uptake in microdissected rat CCT exposed acutely to elevated ambient K+ in vivo and in vitro. Tubules preincubated in 10 mM K+ had higher 86Rb+ uptake than when preincubated in 5 mM K+ (25.9 +/- 1.2 vs. 18.9 +/- 0.7 pmol.mm-1.min-1, P less than 0.001). KCl infusion (5 mumol.100 g-1.min-1 x 60 min) increased 86Rb+ uptake from 19.2 +/- 1.0 to 31.2 +/- 1.4 pmol.mm-1.min-1, P less than 0.001; the increment was preserved in tubules subsequently treated with monensin or nystatin in vitro, suggesting that pump stimulation was not mediated by increased cell Na+. This conclusion was confirmed in separate experiments in which the effect of K+ on 86Rb+ uptake was not altered by concurrent preincubation with amiloride. Studies with CCT from isolated perfused kidneys and from adrenalectomized animals revealed that stimulation of 86Rb+ uptake by a K+ load occurs rapidly (less than or equal to 5 min) and is independent of hormonal factors. Increased external K+ produces a rapid rise in K+-transporting capacity (turnover rate) of the Na+-K+ pump in CCT. This phenomenon probably represents a direct effect on K+ on the pump and is an important component of the early renal response to increased K+ secretory load.

Effects of calcium on ADH action in the cortical collecting tubule perfused in vitro

1982 ◽  
Vol 243 (5) ◽  
pp. F481-F486
Author(s):  
S. Goldfarb
Keyword(s):  
Hydraulic Conductivity ◽  
Water Flow ◽  
Collecting Tubule ◽  
Bath Water ◽  
Mammalian System ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

To test the effects of calcium on ADH action in an in vitro mammalian system, the rabbit cortical collecting tubule was studied. After 25 microunits/ml ADH (n=8) in the presence of 1.25 mM calcium bath, water flow (Jv) rose to 1.56 +/- 0.34 nl.mm-1. min-1 and hydraulic conductivity (Lp, cm.s-1.atm-1 X 10(7)) rose to 123 +/- 22. After 25 microunits/ml ADH in the presence of 3.75 mM calcium bath (n=7), Jv rose to 2.96 +/- 0.6 nl.mm-1.min-1 (P less than 0.05 vs. control) and Lp rose to 286 +/- 62 cm.s-1.atm-1 X 10(7) (P less than 0.02 vs. 1.25 mM bath calcium control). Tubules (n=6) perfused with 3.75 mM Ca and bathed in 3.75 mM Ca also showed an Lp of 279 +/- 82 cm.s-1.atm-1 X 10 (7) following 25 microunits/ml ADH. Tubules similarly studied in 1.25 (n=6) or 3.75 mM Ca (n=6) bath but treated with 10(-4) M 8-[p-chlorophenylthio]cAMP demonstrated Lp of 222 +/- 26 and 235 +/- 37 cm.s-1.atm-1 X 10(7), respectively. These data suggest that increased bath Ca enhances ADH- but not cAMP-stimulated water flow in the rabbit cortical collecting tubule. High perfusate Ca2+ does not alter the stimulatory effect of elevated peritubular Ca2+.

Use of fluorescent dye BCECF to measure intracellular pH in cortical collecting tubule

1989 ◽  
Vol 256 (5) ◽  
pp. F957-F964 ◽  
Author(s):  
I. D. Weiner ◽  
L. L. Hamm
Keyword(s):  
Intracellular Ph ◽  
Bathing Solution ◽  
Intercalated Cells ◽  
Acetoxymethyl Ester ◽  
Collecting Tubule ◽  
Luminal Perfusion ◽  
Apparent Decrease ◽  
Continuous Excitation ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

The compound 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) is being extensively used to measure intracellular pH (pHi) in a variety of tissue preparations. Use of this dye in the isolated perfused rabbit cortical collecting tubule (CCT) revealed two previously unreported findings. Apical incubation via luminal perfusion with the acetoxymethyl ester of BCECF (BCECF-AM) resulted in intense uptake into a minority cell population. Basolateral incubation via the peritubular bathing solution resulted in apparent homogenous uptake into all cells. The minority cells were identified as intercalated cells by the use of fluorescent probes specific for intercalated cells. Minority cell pHi was 7.34 +/- 0.06 (n = 9); majority cell pHi was 7.41 +/- 0.05 (n = 6). In addition, prolonged excitation of BCECF resulted in adverse effects. Intense continuous excitation for 1-3 min resulted in a fall in apparent pHi of 0.53 +/- 0.05 pH units (n = 6, P less than 0.001). After reloading with fresh BCECF-AM, the calculated pHi was 0.05 +/- 0.07 pH units higher than before prolonged excitation (P = NS), suggesting that the apparent decrease was due to changes in the pH sensitivity of BCECF as a result of the prolonged excitation. Prolonged excitation of cells with pHi clamped at 7.50 resulted in a decline in apparent pHi at the rate of 0.15 +/- 0.02 pH units/min (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Cell volume regulation of rabbit cortical collecting tubule in anisotonic media

1990 ◽  
Vol 258 (6) ◽  
pp. F1657-F1665 ◽  
Author(s):  
E. Natke
Keyword(s):  
Volume Regulation ◽  
Control Volume ◽  
Cell Volume Regulation ◽  
Volume Regulatory Decrease ◽  
Collecting Tubule ◽  
Collecting Tubules ◽  
Volume Regulatory Increase ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

Volume regulation of nonperfused rabbit cortical collecting tubules in anisotonic bathing media was examined in vitro. When media osmolality is abruptly increased by 150 mosmol/kgH2O with the addition of NaCl, tubules shrink by 20% but do not volume regulate. However, volume regulatory increase (VRI) is observed when 1 mM butyrate is present in the bathing media or when tubules are pretreated with hypotonic media. When media osmolality is increased, butyrate-treated tubules shrink to 74% of their isotonic control volume. As evidence of volume regulation, butyrate-treated tubules swell while still bathed in hypertonic media, recovering in 30 min 78% of the volume lost due to osmotic shrinkage. The butyrate effect requires external Na+ and is inhibited by amiloride. When media osmolality is lowered to 150 mosmol/kgH2O, nonbutyrate tubules swell before showing typical volume regulatory decrease. When these tubules are returned to isotonic media, they immediately shrink to 78% of control volume before showing evidence of VRI. These results suggest that, under the appropriate conditions, cortical collecting tubules are capable of VRI.

Pathways of K+ permeation across the rabbit cortical collecting tubule: effect of amiloride

1984 ◽  
Vol 246 (4) ◽  
pp. F457-F466 ◽  
Author(s):  
J. B. Stokes
Keyword(s):  
Rate Coefficient ◽  
Single File ◽  
Collecting Tubule ◽  
Before And After ◽  
Transepithelial Voltage ◽  
Diffusion Voltage ◽  
K Permeability ◽  
K Transport ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

These experiments were designed to examine passive K+ transport by the rabbit cortical collecting tubule. Potassium diffusion voltages were used to assess the presence of apical and basolateral K+ conductances. With amiloride (0.1 mM) in the lumen, reproducible K+ diffusion voltages from both the lumen and bath were obtained. Amiloride enhanced the magnitude of these voltage deflections (delta VT). There were time-dependent increases in the K+ diffusion voltage but the steady-state values were highly reproducible in the same tubule. In the amiloride-treated tubules, delta VT induced by raising bath [K+] to 20 mM was larger than that produced by the same increase in lumen [K+]. To evaluate whether the amiloride-treated tubule had, as suggested by the K+ diffusion voltages, substantial K+ permeabilities on both apical and basolateral membranes, the K+ rate coefficient (lumen-to-bath, KK) was measured before and after amiloride treatment. The amiloride-induced increase in KK, from 66 +/- 6 to 205 +/- 35 nm/s, was significantly larger than could be accounted for by the changes in transepithelial voltage or membrane voltages alone. This discrepancy could be due to single-file diffusion across the apical membrane and/or the (secondary) enhancement of K+ permeability following inhibition of Na+ transport.

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