PARTIAL RECOVERY OF IN-VIVO FUNCTION BY IMPROVED INCUBATION CONDITIONS OF ISOLATED RENAL PROXIMAL TUBULE .2. CHANGE OF NA-HCO3 COTRANSPORT STOICHIOMETRY AND OF RESPONSE TO ACETAZOLAMIDE

In the preceding publication we reported that some transport propertie s of proximal tubules perfused in vitro differ from those of tubules p erfused in vivo, and that the in vivo function can be largely recupera ted by improved metabolic substrate supply and stimulation with norepi nephrine (NE). Since we have previously observed that the basolateral Na-HCO3-cotransporter operates with an overall stoichiometric ratio of q approximate to 3 HCO3- :1 Na+ in vivo, but with q approximate to 2 HCO3- :1 Na+ in vitro and that it responds differently in both cases t o acetazolamide (ACZ), we have now tested whether the cotransporter ca n regain its in vivo function in vitro if the incubation conditions ar e improved. Cell membrane potentials (Vb) and cell pH (pH(i)) were mea sured with microelectrodes and microfluorimetric techniques on isolate d S2 segments of rabbit proximal tubule and the instantaneous V-b resp onse to a 2: 1 reduction of bath HCO3- or Na+ concentration was determ inded. (Delta V-b)HCO3- and (Delta V-b)Na averaged 13.1 +/- 0.9 mV (SE M) and 6.9 +/- 0.5 mV in D-glucose-containing control HCO3- Ringer sol ution 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 Ci increased from 1.9 to 2.7. Concomitantly the tubules became susceptible to ACZ (1 mmol/l), which reduced (Delt a V-b)HCO3 in control conditions only to 94.6 +/- 1.2% but under impro ved incubation conditions to 64.5 +/- 2.4%. As verified in voltage div ider measurements the latter reduction was not caused by activation of a basolateral K+ conductance. The results indicate that improved incu bation conditions can at least partially revert cotransport function t owards that of the in vivo state. The effect of ACZ may be explained i f in the improved state 1 CO32- + 1 HCO3- + 1 Na+ are cotransported, i n which case inhibition of carbonic anhydrase (CA) may cause a CO32-/p H disequilibrium to develop in the basal labyrinth which impedes the c otransport. Under conventional incubation conditions, however, when on ly 2 HCO3- + 1 Na+ are cotransported no such disequilibrium 3 should d evelop irrespective of whether CA is active or inhibited.