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 HCO(3-)-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 approximately =40%, and in the presence of DMEM or RPMI 1640 it increased J(HCO3) by approximately =100%. This stimulation by NE followed Michaelis-Menten kinetics with an EC50 value of 0.25 micromol/l and was probably mediated by alpha1-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.