PARTIAL RECOVERY OF IN-VIVO FUNCTION BY IMPROVED INCUBATION CONDITIONS OF ISOLATED RENAL PROXIMAL TUBULE .1. CHANGE OF AMILORIDE-INHIBITABLE K+ CONDUCTANCE

Isolated microperfused rabbit renal proximal tubule S2 segments, if in cubated in conventional substrate containing HCO3- Ringer solution, ex hibit lower cell membrane potentials (V-b) and elevated intracellular Na+ concentrations ([Na](i)) compared to rat tubules in vivo. Assuming that these and other differences reflect insufficient metabolic and/o r hormonal stimulation of the cells, we have used microelectrode techn iques to test whether improving substrate supply and applying norepine phrine (NE, to compensate for the missing nerve supply) reverts V-b an d [Na](i) to values observed in vivo. Application of D-glucose (5.5 mm ol/l) and additional application of pyruvate, lactate, or L-alanine (e ach 10 mmol/l), or bathing the tubules in Dulbecco's modified Eagle's tissue culture medium (DMEM) significantly increased V-b and, whenever tested, reduced [Na](i) as compared to substrate-free or D-glucose-co ntaining control solution and these effects could be prevented - as te sted in the case of pyruvate - by inhibition of the Na/K pump with oua bain. However, high concentrations of acetate, beta-hydroxybutyrate, o r L-glutamine had no significant effect. The largest effect was obtain ed with joint application of DMEM and NE (10 mu mol/l) which increased V-b from -42.8 +/- 1.3 mV (SEM) to -55.3 +/- 2.5 mV (n = 11). Interes tingly we noticed that under the latter conditions the V-b response to bath application of 1 mmol/l amiloride virtually disappeared, i.e. it changed from a depolarization of +14.6 +/- 1.4 mV (in D-glucose Ringe r solution) to +0.6 +/- 0.7 mV (in DMEM plus NE) (n = 8), with some tu bules showing even a small hyperpolarization. The latter implies parti al restoration of the in vivo behaviour, since in experiments on rat p roximal tubules in vivo amiloride regularly hyperpolarized the cells ( by -3.4 +/- 0.76 mV, n = 5). Obviously under conventional in vitro con ditions an amiloride-inhibitable K+ conductance is activated which is inactive in vivo and also inactivates under improved conditions in vit ro. In agreement with observations reported in the subsequent publicat ion our results demonstrate that isolated proximal tubules undergo fun ctional alterations which may be largely prevented by improved metabol ic and stimulatory incubation conditions.