Fig. 8. Glutamine is produced and released by Müller cells exposed simultaneously to glutamate and NH4Cl.A, B, Measurements of [NH4+] by a fiber-optic ammonia sensor. To optimize the detection of ammonia, the pH of the bathing solution in this type of experiment was adjusted to 7.8.A, Control experiment. When the sensor was in the bath, addition of neutral glutaminase (100 μl, in Ringer's solution at pH 7.8, added to 2 ml bath; final activity: 0.01 U/ml) did not produce an increase in the [NH4+] signal, but a decrease caused by dilution, indicating that the glutaminase solution contained much less than 10 μmNH4+. B, When the sensor was positioned close to freshly isolated Müller cells forming a microisland (Fig. 7A), and after cells were exposed to glutamate and NH4+, addition of glutaminase caused a large increase in [NH4+]. Twenty minutes before the beginning of this record, 5 μm NH4Cl was added to the Ringer's solution bathing the cells. As shown, further addition of 50 μm glutamate caused a rapid drop of [NH4+] near the cells, indicating substantial uptake of ammonia by the cells. The time constant of this drop was similar to the time constant of the sensor response. After 10 min of exposing the cells to glutamate and NH4+, glutaminase was added to the bath, causing the rapid hydrolysis of glutamine present in the bath and a concomitant increase of [NH4+].C, Amounts of glutamine (Gln, filled triangles) and alanine (Ala, open circles) released into the bath by a microisland of ∼850 Müller cells after 30 min incubating periods in normal Ringer's solution (all bath collections except third) or in Ringer's solution containing glutamate and NH4Cl (50 μm each, third bath collection). Exposure to glutamate and NH4Cl caused a sizable increase in the production and release of glutamine and alanine. Amino acid content of bath collections was measured by HPLC, using ortho-phthalaldehyde derivatization.