Incubation in tissue culture media allows isolated rabbit proximal tubules to regain in-vivo-like transport function: response of HCO₃ ^– absorption to norepinephrine

Abstract.

Using a new stop-flow perfusion technique with microspectrofluorometric determination of luminal fluid pH, we have studied which substrates or incubation conditions allow isolated rabbit proximal tubules to attain in-vivo-like rates of HCO₃ ^– absorption (J _HCO3) and maximal responses of J _HCO3 to norepinephrine (NE). Essentially three incubation media were tested: plasma-like HCO₃ ^–-Ringer solution containing 5 mmol/l d-glucose (G-Ringer sol.), the same solution also containing 10 mmol/l lactate and 5 mmol/l l-alanine, (LAG-Ringer sol.), and two tissue culture media (DMEM and RPMI 1640). Compared to G-Ringer sol., application of LAG-Ringer sol. in the bath and/or lumen, or application of DMEM or RPMI 1640 in the bath either slightly increased or decreased J _HCO3 with borderline significance. However, RPMI 1640 plus 1 mmol/l pyruvate stimulated J _HCO3 by 55%. While NE (10^–5 mol/l), if applied in G-Ringer sol., had no effect, in the presence of LAG-Ringer sol. it increased J _HCO3 by ≅40%, and in the presence of DMEM or RPMI 1640 it increased J _HCO3 by ≅100%. This stimulation by NE followed Michaelis–Menten kinetics with an EC₅₀ value of 0.25 µmol/l and was probably mediated by α₁-adrenergic receptors. Additional cell pH measurements suggest that NE stimulates the basolateral Na⁺-HCO₃ ^– cotransporter which then becomes susceptible to inhibition by cAMP. We conclude that incubation in tissue culture media allows isolated proximal tubules to maintain a better functional state than the commonly used solutions with unphysiologically high substrate concentrations.