Techniques were developed for the measurement of intracellular potentials and potassium activities in rat proximal tubule cells using double barreled K+ liquid-ion-exchanger microelectrodes. After obtaining measurements of stable and reliable control values, the effects of K+ depletion and metabolic and respiratory acidosis on the intracellular potential and K+ activity in rat kidney proximal tubular cells were determined. At a peritubular membrane potential of -66.3 +/- 1.3 mV (mean +/- SE), intracellular K+ activity was 65.9 +/- 2.0 mEq/liter in the control rats. In metabolic acidosis [70 mg NH4Cl/100 g body wt) the peritubular membrane potential was significantly reduced to -47.5 +/- 1.9 mV, and cellular K+ activity to 53.5 +/- 2.0 mEq/liter. In contrast, in respiratory acidosis (15% CO2) the peritubular membrane potential was significantly lowered to -46.1 +/- 1.39 mV, but the cellular K+ activity was maintained at an almost unchanged level of 63.7 +/- 1.9 mEq/liter. In K+ depleted animals (6 weeks on low K+ diet), the peritubular membrane potential was significantly higher than in control animals, -74.8 +/- 2.1 mV, and cellular K+ activity was moderately but significantly reduced to 58.1 +/- 2.7 mEq/liter, Under all conditions studied, cellular K+ was above electrochemical equilibrium. Consequently, an active mechanism for cellular K+ accumulation must exist at one or both cell membranes. Furthermore, peritubular HCO3- appears to be an important factor in maintaining normal K+ distribution across the basolateral cell membrane.
Isolated, polarized, proximal tubule cells of Rana pipiens were voltage clamped and examined for both single-channel and whole cell currents. Barium-sensitive whole cell conductances were calculated from the difference in slopes of the current-voltage relations before and after 5 mM external barium. In 11 voltage-clamped cells with high K in the pipette (and cell), isosmotic addition of 40 mM glucose to the bathing solution increased cell volume by 23 +/- 4% within 2-3 min and increased barium-sensitive conductance by 40 +/- 10% from 0.5 to 0.7 nS (P < 0.005, with each cell as its own control). Isosmotic addition of nonmetabolizable methyl-alpha-D-glucopyranoside, which enters with Na across the apical membrane, produced a similar increase in barium-sensitive conductance (30 +/- 13%). In contrast, 3-O-methyl-D-glucopyranose, which is not cotransported with Na, did not alter either cell volume or barium-sensitive conductance. Isosmotic addition of 40 mM phenylalanine (Phe) increased cell volume by 21 +/- 3% and increased barium-sensitive conductance by 36 +/- 19% from 1.1 to 1.5 nS (P < 0.005, with each cell as its own control; n = 8). All K channels observed at the basolateral membrane of these amphibian cells were found to be activated by pipette suction (stretch) and inhibited by 5 mM external barium (outside-out patches). Hence, stretch-activated (SA) K channels must be mediating the macroscopic increase in whole cell K conductance (GK) after isosmotic addition of glucose and Phe. The process does not seem to involve changes in ATP, because Phe increased GK even more when cytosolic ATP was maintained at high levels (10(-4) M extracellular ouabain and 5 mM intracellular ATP). It is also unlikely that changes in cell pH or calcium mediate the increase in GK, because the bulk composition of the cell is "clamped" by the pipette solution in these experiments (1-micron tip patch pipettes). Consequently, the substrate-induced increase in GK probably arises from a swelling-associated deformation of the submembane cytoskeleton or a direct change in membrane tension. In either case, SA channels would play a physiological role in proximal tubule K homeostasis during sugar and amino acid reabsorption in the proximal tubule of the kidney.
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