Abstract
Primary cultures of rat renal inner medullary collecting duct cells were grown to confluence on glass coverslips and treated permeant supports, and the pH-sensitive fluorescent probe 2,7-biscarboxyethyl-5,6-carboxyfluorescein was employed to delineate the nature of the transport pathways that allowed for recovery from an imposed acid load in a HCO3-/CO2-buffered solution. The H+ efflux rate of acid-loaded cells was 13.44 +/- 0.94 mM/min. Addition of amiloride, 10(-4) M, to the recovery solution reduced the H+ efflux rate to 4.06 +/- 0.63 mM/min. The amiloride-resistant pHi recovery mechanism displayed an absolute requirement for Na+ but was Cl(-)-independent. Studies performed on permeable supports demonstrated that the latter pathway was located primarily on the basolateral-equivalent (BE) cell surface and was inhibited by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In a Na(+)-replete solution containing DIDS (50 microM) and amiloride (10(-4) M), acid-loaded cells failed to return to basal pHi. To delineate further the amiloride-inhibitable component of pHi recovery, monolayers were studied in the nominal absence of HCO3-/CO2. In 70% of monolayers studied, Na(+)-dependent, amiloride-inhibitable H+ efflux was the sole mechanism whereby acid-loaded cells returned to basal pHi. A Na(+)-independent pathway was observed in 30% of monolayers examined and represented only a minor component of the pHi recovery process. In studies performed on permeable supports, the Na(+)-dependent amiloride-inhibitable pathway was found to be confined exclusively to the BE cell surface. In summary, confluent monolayers of rat renal inner medullary collecting duct cells in primary culture possess two major mechanisms that contribute toward recovery from an imposed acid load, namely, Na+/H+ antiport and Na+/HCO3- cotransport. Na(+)-independent pHi recovery mechanisms represent a minor component of the pHi recovery process in the cultured cell. Both the Na+/H+ antiporter and Na+/HCO3- cotransporter are located primarily on the BE cell surface.
Highlights
Primary cultures of rat renal inner medullary col- pH
Delineation of the Polar Location of the Na+/HCO: Cotransporter-Primary cultures of inner medullary collecting duct (IMCD) cells grown to confluence on transparent membranes were used to delineate whether the C1"independent Na+/HCO, transport pathway was of LE or BE location
The results presented demonstrate that rat IMCD cells in primary culture recover from an imposed acid load viaat least two mechanisms, namely amiloride-inhibitable Na+/H+ antiport (Figs. 2, 3, and 10) and DIDS-inhibitable, C1"independent, Na+/HCO, cotransport (Figs. 4-10)
Summary
Compositionof solutions used vers proceeded as described above for coverslip experiments. The final osmolarity of all solutions was adjusted to 301 mOSM. In experiments where gluconate was the substitutedanion, the Ca2+. The pH of all solutions was adjusted to 7.4. ABCDEFGH ble-barreled Harvard infusion pump enabled differential perfusion of the two compartments at thesame flow rate. Correlation of Fluorescence Intensity with pH,-To correlate excitation ratio 500/440 with pH,, a calibration curve was constructed using the high K+/nigericin technique previously described [2]. 2 2 2 linear function of excitation ratio 500/440 over the pH, range 7.5-6.0. Calibration curves obtained on glass coverslips,y = 1.472 - 6.69 ( r = 0.991, n = 114), and on permeable supports, y = 0.702 - 2.16
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