Na–Pi cotransporter type I activity causes a transient intracellular alkalinization during ATP depletion in rabbit medullary thick ascending limb cells

The cellular pathophysiology of renal ischemia–reperfusion injury was investigated in primary cell cultures from rabbit medullary thick ascending limb (MTAL). Metabolic inhibition (MI) was achieved with cyanide and 2-deoxyglucose. Sixty minutes of MI caused a profound but reversible decrease in intracellular concentration of ATP ([ATP]i). Intracellular pH (pHi) first decreased after initiation of MI, followed by a transient alkalinization. When [ATP]i reached its lowest value (<1% of control), the cells slowly acidified to reach a stable pHi of 6.92 after 50 min of MI. In the presence of EIPA (10 µmol/L), the pattern of changes in pHi was unchanged and acidification was not increased, indicating that the Na+/H+ exchangers were inactive during ATP depletion. When inorganic phosphate (Pi) or Na+ was omitted from the apical solutions during MI, the transient alkalinization was no longer observed and the cytosol slowly acidified. Experiments on Na+-dependent alkalinizations revealed the presence of a Na–Pi cotransporter in the apical cell membrane. With indirect immunofluorescence, the Na–Pi cotransporter expressed in these primary cell cultures could be identified as Na–Pi type I. Although the exact physiological role of Na–Pi type I still is unresolved, these experiments demonstrate that apical Na–Pi type I activity is increased at the onset of ATP depletion in MTAL cells.

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Ca2+-activated K+ channels in cultured medullary thick ascending limb cells

AJP Cell Physiology ◽

10.1152/ajpcell.1987.252.2.c121 ◽

1987 ◽

Vol 252 (2) ◽

pp. C121-C127 ◽

Cited By ~ 50

Author(s):

S. E. Guggino ◽

W. B. Guggino ◽

N. Green ◽

B. Sacktor

Keyword(s):

Cell Membrane ◽

Single Channel ◽

Apical Cell ◽

Outward Current ◽

K Channel ◽

Thick Ascending Limb ◽

Patch Clamp Technique ◽

Apical Cell Membrane ◽

K Channels ◽

Medullary Thick Ascending Limb

The conductive properties of a clone of medullary thick ascending limb (MTAL) cells (GRB-MAL1) were assessed using conventional microelectrodes and the patch clamp technique. The apical cell membrane potential (Va) of MTAL cells was -46 +/- 3 mV. Addition of Ba2+ (1 mM) to the apical solution induced a 22 +/- 2 mV depolarization of Va, whereas furosemide hyperpolarized Va by -5 +/- 1 mV. In the cell-attached patch configuration, the most frequently occurring channel had a single channel conductance of 121 +/- 5 pS and carried outward current. In excised patches, current movement was down the electrochemical K+ gradient. Fluctuations were activated by depolarization of Va and by increasing Ca2+ concentration on the intracellular face. Micromolar amounts of Ba2+ on the intracellular face of the membrane inhibited channel activity. We conclude that cultures of MTAL cells GRB-MAL1 retain at least two of the properties of the mature phenotype, namely, an apical K+ conductance and a sensitivity to loop diuretics; the most frequently occurring channel in the apical cell membrane is a Ca2+-activated, maxi-K+ channel; and, finally Ca2+-activated K+ channels may play a role in generating the apical K+ conductance in cultured MTAL cells.

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Membrane stretch: a physiological stimulator of Ca2+-activated K+ channels in thick ascending limb

AJP Renal Physiology ◽

10.1152/ajprenal.1989.257.3.f347 ◽

1989 ◽

Vol 257 (3) ◽

pp. F347-F352 ◽

Cited By ~ 22

Author(s):

J. Taniguchi ◽

W. B. Guggino

Keyword(s):

Negative Pressure ◽

Apical Cell ◽

Bathing Solution ◽

Thick Ascending Limb ◽

Open Probability ◽

Apical Cell Membrane ◽

K Channels ◽

Membrane Stretch ◽

Medullary Thick Ascending Limb ◽

Tubular Flow

The effects of membrane stretch on Ca2+-activated (maxi) K+ channels were examined in the apical membrane of cultured medullary thick ascending limb (MTAL) cells. Using cell-attached patchclamp technique, we found that negative pressure (-33 +/- 5 cmH2O) applied to the patch membrane increased fractional open probability (NPo) from 0.3 +/- 0.2 to 29.9 +/- 7.6% (n = 12) in the presence of 1.8 mM Ca2+ in the pipette. The activity returned to control on releasing the negative pressure. Reduction of extracellular osmolality from 293.2 +/- 1.6 to 219.8 +/- 1.1 mosmol/kg also activated K+ channels (NPo = 43.8 +/- 12.2%, n = 8) in cell-attached patches. Removal of Ca2+ from both pipette and bathing solution inhibited osmotic activation of K+ channels. K+ channels were shown to be Ca2+-activated K+ channels by their conductance (146 +/- 7 pS, n = 5) and Ca2+ dependence. Our data suggest that membrane stretch caused by swelling or possibly by tubular flow enhances Ca2+ entry across the apical cell membrane of MTAL cells activating maxi K+ channels.

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Blocking agents of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells

AJP Cell Physiology ◽

10.1152/ajpcell.1987.252.2.c128 ◽

1987 ◽

Vol 252 (2) ◽

pp. C128-C137 ◽

Cited By ~ 73

Author(s):

S. E. Guggino ◽

W. B. Guggino ◽

N. Green ◽

B. Sacktor

Keyword(s):

Single Channel ◽

Apical Cell ◽

Thick Ascending Limb ◽

Extracellular Solution ◽

Apical Cell Membrane ◽

K Channels ◽

Blocking Agents ◽

Medullary Thick Ascending Limb ◽

Open Time ◽

Voltage Dependent

Ca2+-activated K+ channels with estimated single channel conductances of 127 +/- 2 pS were identified in the apical cell membrane of clone A3 of cultured medullary thick ascending limb (MTAL) cells. Both Ba2+ and the scorpion toxin, charybdotoxin (CTX), are slow blockers of the channels. An application of 0.1 microM Ba2+ to the intracellular face caused a 50% reduction in fractional open time (fv). Ba2+ block is both concentration and voltage dependent. Concentrations of CTX as low as 2 nM in the extracellular solution caused a significant reduction in fv. Tetraethylammonium (TEA) and quinine are fast blockers of Ca2+-activated K+ channels in MTAL cells. TEA, 400 microM, in the extracellular solution caused a voltage-dependent reduction in channel amplitude, whereas it takes 10 mM in the intracellular solution to reduce channel amplitude by 30%. Micromolar amounts of quinine applied to the intracellular face caused the channels to flicker rapidly between open and blocked states. These results suggest that K+ channels in MTAL cells are homologous to those found in muscle cells, and that these blocking agents may be used to probe the nature of K+ conductances in several nephron segments.

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Active absorption of NH4+ by rat medullary thick ascending limb: inhibition by potassium

AJP Renal Physiology ◽

10.1152/ajprenal.1988.255.1.f78 ◽

1988 ◽

Vol 255 (1) ◽

pp. F78-F87 ◽

Cited By ~ 11

Author(s):

D. W. Good

Keyword(s):

Potassium Concentration ◽

Apical Cell ◽

Thick Ascending Limb ◽

Apical Cell Membrane ◽

Active Absorption ◽

Medullary Thick Ascending Limb ◽

Total Ammonia ◽

Cotransport System ◽

Ammonia Absorption

These experiments were designed to determine the relative contributions of active NH4+ transport and voltage-driven NH4+ diffusion to direct NH4+ absorption by the medullary thick ascending limb of the rat. Medullary thick ascending limbs were perfused in vitro with solutions containing 25 mM HCO3 and 4 mM total ammonia. Under steady-state conditions, the lumen-positive transepithelial voltage (VT) was not sufficient to account for the observed decrease in lumen NH4+ concentration, consistent with active absorption of NH4+. Flux calculations based on VT and measured NH4+ permeability (6 x 10(-5) cm/s) indicate that the majority (at least 65%) of total ammonia absorption is due to active transport of NH4+. The remainder of NH4+ absorption can be accounted for by voltage-driven diffusion. Increasing the potassium concentration from 4 to 24 mM in perfusate and bath markedly inhibited total ammonia absorption but did not affect VT, NH4+ permeability, or HCO3 absorption. These results are consistent with inhibition of the active component of NH4+ absorption by potassium. The active NH4+ absorption is likely mediated by cotransport of Na+, NH4+, and Cl- across the apical cell membrane. Inhibition of active NH4+ absorption by an increase in potassium concentration may be due, in part, to competition between NH4+ and K+ for a common binding site on the Na+ -K+ -2Cl- cotransport system.

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