ACTIVATION OF NA-2CL-K COTRANSPORT BY LUMINAL CHLORIDE IN MACULA DENSA CELLS

Changes in macula densa intracellular pH (pH(i)) were used to monitor the direction of flux mediated by the apical Na:2Cl:K cotransporter. A t the macula densa, a decrease in luminal [Cl] ([Cl](I)) from 60 to 1 mM produced cellular alkalinization secondary to a cascade of events i nvolving a decrease in apical Na:2Cl:K cotransport, a fall in intracel lular [Na] ([Na](i)) and a stimulation of Na:H exchange. This is suppo rted by the fact that 97% of the change in macula densa pH(i) with red uction in [Cl](l) was bumetanide-sensitive whereas 92% of this pH chan ge was amiloride-sensitive. We found that, in the presence of 20 mM Na and 5 mM K, a [Cl](l) of 14.3 +/- 2.4 mM (N = 7) produced equilibrium of the apical cotransporter since the pH(i) obtained under this condi tion was identical to the pH, found after reducing the net ionic flux to zero with bumetanide. Using this Value together with the expected s toichiometry for the bumetanide-sensitive cotransporter, it was estima ted that the intracellular [CI] ([C](i)) at equilibrium (or in the pre sence of bumetanide) could be as low as 5 mM. Also, using a Hill numbe r of 2 which is consistent with the present data, the affinity for [Cl ](l) was found to be 32.5 mM. Under physiological luminal conditions p revailing at the end of the thick ascending limb (similar to 3.5 mM K, and similar to 25 to 30 mM NaCl), macula densa cells are probably ope rating close to equilibrium while maintaining a small net reabsorption of Na/K and Cl. Since macula densa cells appear capable of reducing [ Cl](i) to very low levels, a reabsorptive flux should continue to occu r until [NaCl](l) is reduced to 18 mM.