Effect of cell swelling, NH4Cl and glutamate on acridine orange fluorescence in retinal ganglion cells

Acridine orange has been utilized to test for an effect of cell swelling and glutamate on pH in acidic intracellular compartments of retinal ganglion cells. Alkalinization of these compartments is reflected by an increase in acridine orange fluorescence intensity at >530 nm. NH4Cl (500 mu M) and chloroquine (100 mu M), weak bases known to alkalinize acidic compartments, expectedly increased acridine orange fluorescence. A similar effect was elicited by a decrease in extracellular osmolarity. An increase in extracellular K+ concentration, leading to isotonic cell swelling, also increased acridine orange fluorescence. Addition of 100 mu M glutamate increased acridine orange fluorescence, although it did not appear to affect cell volume. The glutamate-induced alkalinization was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, and mimicked by NMDA (50 mu M). The effect of NMDA was blocked by the removal of extracellular Ca2+. Similarly, NMDA-induced increases in intracellular Ca2+ were blocked under Ca2+-free conditions. Cytosolic pH measurements in the same cells demonstrated an acidification in response to reduced extracellular osmolarity. In contrast, a high K+ solution and 100 mu M glutamate both increased cytosolic pH. In conclusion, alkalinization of acidic cellular compartments of retinal ganglion cells is induced not only by NH4Cl, chloroquine and maneuvers which induce cell swelling, but also by the neurotransmitter glutamate. The mechanism by which glutamate induces its effect appears to be primarily via increases in intracellular Ca2+. The alkalinization of acidic intracellular compartments, as such, may modify transmitter metabolism and trafficking of cell membrane proteins.