Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule
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In the preceding publication we reported that some transport properties of proximal tubules perfused in vitro differ from those of tubules perfused in vivo, and that the in vivo function can be largely recuperated by improved metabolic substrate supply and stimulation with norepinephrine (NE). Since we have previously observed that the basolateral Na-HCO3-cotransporter operates with an overall stoichiometric ratio of q ≈3 HCO3– :1 Na+ in vivo, but with q ≈2 HCO3–:1 Na+ in vitro and that it responds differently in both cases to acetazolamide (ACZ), we have now tested whether the cotransporter can regain its in vivo function in vitro if the incubation conditions are improved. Cell membrane potentials (Vb) and cell pH (pHi) were measured with microelectrodes and microfluorimetric techniques on isolated S2 segments of rabbit proximal tubule and the instantaneous Vb response to a 2:1 reduction of bath HCO3–or Na+ concentration was determinded. (ΔVb)HCO3 and (ΔVb)Na averaged 13.1 ± 0.9 mV (SEM) and 6.9 ± 0.5 mV in D-glucose-containing control HCO3-Ringer solution and decreased respectively to 10.1 ± 0.5 mV and 3.8 ± 0.2 mV (n = 8) after incubation in tissue culture medium and NE (10–5 mmol/l). These data imply that q increased from 1.9 to 2.7. Concomitantly the tubules became susceptible to ACZ (1 mmol/l), which reduced (ΔVb)HCO3 in control conditions only to 94.6 ± 1.2% but under improved incubation conditions to 64.5 ± 2.4%. As verified in voltage divider measurements the latter reduction was not caused by activation of a basolateral K+ conductance. The results indicate that improved incubation conditions can at least partially revert cotransport function towards that of the in vivo state. The effect of ACZ may be explained if in the improved state 1 CO32–+ 1 HCO3–+ 1 Na+ are cotransported, in which case inhibition of carbonic anhydrase (CA) may cause a CO32–/pH disequilibrium to develop in the basal labyrinth which impedes the cotransport. Under conventional incubation conditions, however, when only 2 HCO3–+ 1 Na+ are cotransported no such disequilibrium should develop irrespective of whether CA is active or inhibited.
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