scholarly journals Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3- transport.

1983 ◽  
Vol 81 (1) ◽  
pp. 53-94 ◽  
Author(s):  
W F Boron ◽  
E L Boulpaep
Keyword(s):  
Negative Charge ◽  
Ph Regulation ◽  
Basolateral Membrane ◽  
Proximal Tubules ◽  
Ambystoma Tigrinum ◽  
Intracellular Ph Regulation ◽  
Rapid Fall ◽  
Tight Linkage ◽  
Series Of Experiments ◽  
Basolateral Membrane Potential

We have used pH-, Na-, and Cl-sensitive microelectrodes to study basolateral HCO3- transport in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum. In one series of experiments, we lowered basolateral pH (pHb) from 7.5 to 6.8 by reducing [HCO3-]b from 10 to 2 mM at a constant pCO2. This reduction of pHb and [HCO3-]b causes a large (approximately 0.35), rapid fall in pHi as well as a transient depolarization of the basolateral membrane. Returning pHb and [HCO3-]b to normal has the opposite effects. Similar reductions of luminal pH (pHl) and [HCO3-]l have only minor effects. The reduction of [HCO3-]b and pHb also produces a reversible fall in aiNa. In a second series of experiments, we reduced [Na+]b at constant [HCO3-]b and pHb, and also observed a rapid fall in pHi and a transient basolateral depolarization. These changes are reversed by returning [Na+]b to normal. The effects of altering [Na+]l in the presence of HCO3-, or of altering [Na+]b in the nominal absence of HCO3-, are substantially less. Although the effects on pHi and basolateral membrane potential of altering either [HCO3-]b or [Na+]b are largely blocked by 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonate (SITS), they are not affected by removal of Cl-, nor are there accompanying changes in aiCl consistent with a tight linkage between Cl- fluxes and those of Na+ and HCO3-. The aforementioned changes are apparently mediated by a single transport system, not involving Cl-. We conclude that HCO3- transport is restricted to the basolateral membrane, and that HCO3- fluxes are linked to those of Na+. The data are compatible with an electrogenic Na/HCO3 transporter that carries Na+, HCO3-, and net negative charge in the same direction.

Mechanism of bicarbonate exit across basolateral membrane of rabbit proximal straight tubule

1987 ◽  
Vol 252 (1) ◽  
pp. F11-F18 ◽  
Author(s):  
S. Sasaki ◽  
T. Shiigai ◽  
N. Yoshiyama ◽  
J. Takeuchi
Keyword(s):  
Negative Charge ◽  
Driving Forces ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Exit Mechanism ◽  
Total Replacement ◽  
Straight Tubules ◽  
Proximal Straight Tubule ◽  
Basolateral Membrane Potential

To clarify the mechanism(s) of HCO3- (or related base) transport across the basolateral membrane, rabbit proximal straight tubules were perfused in vitro, and intracellular pH (pHi) and Na+ activity (aiNa) were measured by double-barreled ion-selective microelectrodes. Lowering bath HCO3- from 25 to 5 mM at constant PCO2 depolarized basolateral membrane potential (Vbl), and reduced pHi. Most of these changes were inhibited by adding 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to the bath. Total replacement of bath Na+ with choline also depolarized Vbl and reduced pHi, and these changes were also inhibited by SITS. Reduction in aiNa was observed when bath HCO3- was lowered. Taken together, these findings suggest that HCO3- exists the basolateral membrane with Na+ and negative charge. Calculation of the electrochemical driving forces suggests that the stoichiometry of HCO3-/Na+ must be larger than two for maintaining HCO3- efflux. Total replacement of bath Cl- with isethionate depolarized Vbl gradually and increased pHi slightly, implying the existence of a Cl(-)-related HCO3- exit mechanism. The rate of decrease in pHi induced by lowering bath HCO3- was slightly reduced (20%) by the absence of bath Cl-. Therefore, the importance of Cl(-)-related HCO3- transport is small relative to total basolateral HCO3- exit. Accordingly, these data suggest that most of HCO3- exits the basolateral membrane through the rheogenic Na+/HCO3- cotransport mechanism with a stoichiometry of HCO3-/Na+ of more than two.

Intracellular pH regulation in rabbit S3 proximal tubule: basolateral Cl-HCO3 exchange and Na-HCO3 cotransport

1990 ◽  
Vol 258 (2) ◽  
pp. F371-F381 ◽  
Author(s):  
N. L. Nakhoul ◽  
L. K. Chen ◽  
W. F. Boron
Keyword(s):  
Proximal Tubule ◽  
Intracellular Ph ◽  
Initial Rate ◽  
Ph Regulation ◽  
Basolateral Membrane ◽  
Intracellular Ph Regulation ◽  
S3 Segment ◽  
Absorbance Spectra ◽  
Rule Out

We studied the role of basolateral HCO3- transport in the regulation of intracellular pH (pHi) in the isolated perfused S3 segment of the rabbit proximal tubule. pHi was calculated from absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein. Solutions were normally buffered to pH 7.4 at 37 degrees C with 25 mM HCO3- 5% CO2. pHi fell by approximately 0.17 when luminal [HCO3-] was lowered to 5 mM at fixed PCO2 (i.e., reducing pH to 6.8) but by approximately 0.42 when [HCO3-] in the bath (i.e., basolateral solution) was lowered to 5 mM. The pHi decrease elicited by reducing bath [HCO3-] was substantially reduced by removal of Cl- or Na+, suggesting that components of basolateral HCO3- transport are Cl- and/or Na+ dependent. We tested for the presence of basolateral Cl-HCO3 exchange by removing bath Cl-. This caused pHi to increase by approximately 0.23, with an initial rate of approximately 100 X 10(-4) pH/s. Although the initial rate of this pHi increase was not reduced by removing Na+ bilaterally, it was substantially lowered by the nominal removal of HCO3- from bath and lumen or by the addition of 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) to the bath. The results thus suggest that a Na-independent Cl-HCO3 exchanger is present at the basolateral membrane. We tested for the presence of basolateral Na-HCO3 cotransport by removing bath Na+. This caused pHi to fall reversibly by approximately 0.26 with initial rates of pHi decline and recovery being approximately 30 and approximately 41 X 10(-4) pH/s, respectively. Although the bilateral removal of Cl- had no effect on these rates, the nominal removal of HCO3- or the presence of DIDS substantially slowed the pHi changes. Thus, in addition to a Cl-HCO3 exchanger, the basolateral membrane of the S3 proximal tubule also appears to possess a Na-HCO3 cotransport mechanism. The data do not rule out the possibility of other basolateral HCO3- transporters.

Intracellular potentials in rabbit proximal tubules perfused in vitro

1981 ◽  
Vol 240 (3) ◽  
pp. F200-F210 ◽  
Author(s):  
B. Biagi ◽  
T. Kubota ◽  
M. Sohtell ◽  
G. Giebisch
Keyword(s):  
Membrane Potential ◽  
Basolateral Membrane ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Electrical Measurements ◽  
Proximal Tubular ◽  
The Mean ◽  
Number Of Cells ◽  
Basolateral Membrane Potential

Conventional microelectrodes were used to measure the basolateral membrane potential (VBL) in isolated perfused superficial proximal convoluted (sPCT) and superficial proximal straight (sPST) tubules of the rabbit kidney. Stable recordings for periods up to 2 h can be obtained. The mean +/- SE (n = number of cells) values of VBL were sPCT = -51.0 +/- 1.63 (24) and sPST = -47.0 +/- 0.97 (94) mV. Inhibitors of active transport, ouabain (10(-5) M) and low bath potassium (0.1 mM), caused a significant depolarization of VBL in sPST. In contrast, short-duration bath cooling (10 degrees C) had no significant effect. Removal of luminal glucose caused a larger hyperpolarization in sPCT (-13.9 +/- 1.77 (9) mV) than in sPST (-3.8 +/- 1.02 (5) mV). Removal of luminal glucose and alanine resulted in an even larger hyperpolarization of VBL in sPCT (-19.0 +/- 0.44 (6) mV). Perfusion of the lumen with a solution resembling late proximal tubular fluid in sPST resulted in hyperpolarization of VBL (-4.3 +/- 0.85 (4) mV). Reducing bath pH to 6.7 depolarized VBL (39.9 +/- 1.77 (13) mV). This effect can be associated with a decrease in the relative potassium permeability of the basolateral membrane. These results demonstrate the feasibility of using intracellular electrical measurements to determine both luminal and basolateral membrane characteristics in isolated proximal tubular segments.

Adaptive responses to Na(+)-coupled solute transport and osmotic cell swelling in salamander proximal tubule

1994 ◽  
Vol 267 (3) ◽  
pp. F479-F488
Author(s):  
S. W. Weinstein ◽  
C. Clausen
Keyword(s):  
Membrane Potential ◽  
Proximal Tubule ◽  
Adaptive Response ◽  
Basolateral Membrane ◽  
Proximal Tubules ◽  
Cell Swelling ◽  
Adaptive Responses ◽  
High Concentration ◽  
Luminal Perfusion ◽  
Basolateral Membrane Potential

Measurements of basolateral membrane potential and relative K+ conductance were performed in isolated perfused proximal tubules from Ambystoma. To investigate adaptive increases in basolateral membrane K+ conductance (gK) associated with Na(+)-solute cotransport, measurements were made comparing transport of glucose and alanine, with changes caused by hypotonicity- and solute-induced cell swelling. Luminal perfusion with alanine produced results consistent with an adaptive increase in gK; perfusion with glucose failed to show this response. Hypotonic peritubular solutions also produced results consistent with an adaptive increase in gK, but isosmotic increases of peritubular glucose sufficient to swell the cells failed to produce this. No changes in the responses to luminal perfusion with alanine or glucose were induced by hypotonic peritubular solutions. With a high concentration of glucose in isosmotic peritubular solutions, perfusion of the lumen with glucose now produced results consistent with an adaptive increase in gK. Isosmotic peritubular solutions containing urea produced adaptive changes similar to those observed using hypotonic peritubular solutions, but when glucose was subsequently added to the lumen, no further adaptive response occurred. We conclude that cell swelling alone is insufficient to explain the mechanisms involved in the adaptive responses of gK occurring during Na(+)-solute cotransport in the salamander proximal tubule.

scholarly journals Effect of electroneutral luminal and basolateral lactate transport on intracellular pH in salamander proximal tubules.

1987 ◽  
Vol 90 (6) ◽  
pp. 799-831 ◽  
Author(s):  
A W Siebens ◽  
W F Boron
Keyword(s):  
Intracellular Ph ◽  
Initial Rate ◽  
Basolateral Membrane ◽  
Proximal Tubules ◽  
Tiger Salamander ◽  
Organic Substrates ◽  
Luminal Membrane ◽  
Acid Extrusion ◽  
Intracellular Alkalinization ◽  
Basolateral Membrane Potential

We used microelectrodes to examine the effects of organic substrates, particularly lactate (Lac-), on the intracellular pH (pHi) and basolateral membrane potential (Vbl) in isolated, perfused proximal tubules of the tiger salamander. Exposure of the luminal and basolateral membranes to 3.6 mM Lac- caused pHi to increase by approximately 0.2, opposite to the decrease expected from nonionic diffusion of lactic acid (HLac) into the cell. Addition of Lac- to only the lumen also caused alkalinization, but only if Na+ was present. This alkalinization was not accompanied by immediate Vbl changes, which suggests that it involves luminal, electroneutral Na/Lac cotransport. Addition of Lac- to only the basolateral solution caused pHi to decrease by approximately 0.08. The initial rate of this acidification was a saturable function of [Lac-], was not affected by removal of Na+, and was reversibly reduced by alpha-cyano-4-hydroxycinnamate (CHC). Thus, the pHi decrease induced by basolateral Lac- appears to be due to the basolateral entry of H+ and Lac-, mediated by an H/Lac cotransporter (or a Lac-base exchanger). Our data suggest that this transporter is electroneutral and is not present at the luminal membrane. A key question is how the addition of Lac- to the lumen increases pHi. We found that inhibition of basolateral H/Lac cotransport by basolateral CHC reduced the initial rate of pHi increase caused by luminal Lac-. On the other hand, luminal CHC had no effect on the luminal Lac(-)-induced alkalinization. These data suggest that when Lac- is present in the lumen, it enters the cell from the lumen via electroneutral Na/Lac cotransport and then exists with H+ across the basolateral membrane via electroneutral H/Lac cotransport. The net effect is transepithelial Lac- reabsorption, basolateral acid extrusion, and intracellular alkalinization.

Depolarization-induced alkalinization in proximal tubules. I. Characteristics and dependence on Na+

1989 ◽  
Vol 256 (2) ◽  
pp. F342-F353 ◽  
Author(s):  
A. W. Siebens ◽  
W. F. Boron
Keyword(s):  
Intracellular Ph ◽  
Initial Rate ◽  
Basolateral Membrane ◽  
Ph Sensitive ◽  
Proximal Tubules ◽  
Ambystoma Tigrinum ◽  
Tiger Salamander ◽  
Additive Effects ◽  
Basolateral Membranes ◽  
Dependent Processes

We used intracellular pH-sensitive and voltage microelectrodes to examine the effects of depolarization on intracellular pH (pHi) in isolated perfused proximal tubules from the tiger salamander Ambystoma tigrinum. Tubules were depolarized by raising [K+] in the bath (b) or lumen (l), or by adding Ba2+ (1 mM) to the bath or lumen, always in nominally HCO3-free solutions. Increasing [K+]b from 2.5 to 50 mM caused the basolateral membrane to depolarize by an average of 45 mV, and pHi to increase by 0.23 over 3 min. Similar alkalinization was observed when basolateral Ba2+ (1 mM) was used to depolarize the cell at constant extracellular [K+], suggesting that the alkalinization observed during exposure to elevated [K+]b results from depolarization rather than an increase in [K+]b. The initial rate of depolarization-induced alkalinization (DIA) was proportional to the magnitude of the depolarization, regardless of whether tubules were depolarized by elevated [K+]b, elevated [K+]l, or by basolateral Ba2+. An exception was the initial rate of the alkalinization caused by 1 mM luminal Ba2+, which was more than 10-fold greater than that predicted from the depolarization. The voltage and pHi responses to basolateral Ba2+ were smaller in some tubules than others, as were the responses to elevated [K+]l. Tubules with small responses to 1 mM [Ba2+]b had large responses to 50 mM [K+]l, whereas tubules with large responses to 1 mM [Ba2+]b had small responses to 50 mM [K+]l. This variability can be accounted for by differences in the luminal K+ conductance. The DIA was partially inhibited by removal of Na+ from only the lumen or only the bath, but completely inhibited by bilateral Na+ removal. We conclude that the depolarization-induced alkalinization results from additive effects of Na+-dependent processes at both the luminal and basolateral membranes.

Isolated perfused salamander proximal tubule. II. Monovalent ion replacement and rheogenic transport

1981 ◽  
Vol 241 (5) ◽  
pp. F540-F555 ◽  
Author(s):  
H. Sackin ◽  
E. L. Boulpaep
Keyword(s):  
Basolateral Membrane ◽  
Electrical Potential ◽  
Transport Current ◽  
Proximal Tubules ◽  
Ambystoma Tigrinum ◽  
Ionic Diffusion ◽  
Renal Epithelium ◽  
Monovalent Ion ◽  
Chloride Removal

Early proximal tubules of the salamander kidney (Ambystoma tigrinum) were isolated and perfused in vitro. Transepithelial and basolateral electrical potential differences, transepithelial resistances, and intracellular ionic activities were measured during removal of Na+, K+, or Cl- from the lumen, the bath, or both lumen and bath. The effects of these external ionic replacements are interpreted in terms of an equivalent circuit that represents the renal epithelium as a network of passive ionic resistances, ionic diffusion potentials, and active transport current sources. Results indicate that rheogenic transport across the basolateral membrane is substantially diminished by removal of Na+ from either lumen or bath or by removal of K+ from the bath. On the other hand, bilateral chloride removal produces an increase in transepithelial resistance but almost no change in the calculated rate of basolateral rheogenic transport. This suggests that the source of the basolateral rheogenic ion flux ia a Na-K-ATPae that actively transports an excess of outward Na+ over inward K+.

Electrophysiology of salamander proximal tubule. I. Effects of rapid cooling

1986 ◽  
Vol 251 (2) ◽  
pp. F319-F333
Author(s):  
H. Sackin
Keyword(s):  
Membrane Potential ◽  
Low Temperature ◽  
Proximal Tubule ◽  
Basolateral Membrane ◽  
Bath Temperature ◽  
Ambystoma Tigrinum ◽  
Ionic Selectivity ◽  
Anion Selectivity ◽  
Nacl Concentration ◽  
Basolateral Membrane Potential

The response of the amphibian proximal tubule to a rapid decrease in temperature was studied in isolated perfused tubules of Ambystoma tigrinum. Cooling from 23 to 4 degrees C increased paracellular and cellular electrical resistances by factors of 1.7 and 3.6, respectively, but had virtually no effect on the ionic selectivity of the paracellular pathway. When lumen and bath solutions were maintained identical by rapid tubule perfusion, decreasing bath temperature from 22 to 0 degree C in 400 ms depolarized the transepithelial potential (Vte) from -3.7 +/- 0.3 to -1.1 +/- 0.2 mV and depolarized the basolateral membrane potential (Vbl) from -52 +/- 3 to -45 +/- 3 mV (n = 12). These fast depolarizations were followed by slower depolarizations of both Vte and Vbl that continued throughout the period of low temperature. Only approximately 30% of the initial slow depolarization of Vte at low temperature could be explained by changes in electrical resistance and cell membrane potential. The remaining 70% of this Vte depolarization at low temperature is consistent with equilibration of a hypertonic interspace with isotonic lumen and bath solutions. Given the anion selectivity of Ambystoma proximal tubule, the magnitude of this slow Vte depolarization implies an interspace NaCl concentration 2-5% higher than the NaCl concentration in either the lumen or bath solutions.

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