Chloride-dependent transport of NH4+ into bee retinal glial cells

Mammalian astrocytes convert glutamate to glutamine and bee retinal glial c ells convert pyruvate to alanine. To maintain such amination reactions thes e glial cells may take up NH4+/NH3. We have studied the entry of NH4+/NH3 i nto bundles of glial cells isolated from bee retina by using the fluorescen t dye BCECF to measure pH. Ammonium caused intracellular pH to decrease by a saturable process: the rate of change of pH was maximal for an ammonium c oncentration of about 5 mM. This acidifying response to ammonium was abolis hed by the loop diuretic bumetanide (100 mu M) and by removal of extracellu lar Cl-. These results strongly suggest that ammonium enters the cell by co transport of NH4+ with Cl-. Removal of extracellular Nat did not abolish th e NH4+ induced acidification. The NH4+-induced pH change was unaffected whe n nearly all K+ conductance was blocked with 5 mM Ba2+ showing that NH4+ di d not enter through Ba2+-sensitive ion channels. Application of 2 mM NH4+ l ed to a large increase in total intracellular proton concentration estimate d to exceed 13.5 mEq/L. As the cell membrane appeared to be permeable to NH 3, we suggest that when NH4+ entered the cells, NH3 left, so that protons w ere shuttled into the cell. This shuttle, which was strongly dependent on i nternal and external pH, was quantitatively modelled. In retinal slices, 2 mM NH4+ alkalinized the extracellular space: this alkalinization was reduce d in the absence of bath Cl-. We conclude that NH4+ enters the glial cells in bee retina on a cotransporter with functional similarities to the NH4+(K +)-Cl- cotransporter described in kidney cells.