Extracellular H⁺ fluxes from tiger salamander Müller (glial) cells measured using self-referencing H⁺-selective microelectrodes

Abstract

Self-referencing H-selective electrodes were used to measure extracellular H fluxes from Müller (glial) cells isolated from the tiger salamander retina. A novel chamber enabled stable recordings using H-selective microelectrodes in a self-referencing format using bicarbonate-based buffer solutions. A small basal H flux was observed from the end foot region of quiescent cells bathed in 24 mM bicarbonate-based solutions, and increasing extracellular potassium induced a dose-dependent increase in H flux. Barium at 6 mM also increased H flux. Potassium-induced extracellular acidifications were abolished when bicarbonate was replaced by 1 mM HEPES. The carbonic anhydrase antagonist benzolamide potentiated the potassium-induced extracellular acidification, while 300 μM DIDS, 300 μM SITS, and 30 μM S0859 significantly reduced the response. Potassium-induced extracellular acidifications persisted in solutions lacking extracellular calcium, although potassium-induced changes in intracellular calcium monitored with Oregon Green were abolished. Exchange of external sodium with choline also eliminated the potassium-induced extracellular acidification. Removal of extracellular sodium by itself induced a transient alkalinization, and replacement of sodium induced a transient acidification, both of which were blocked by 300 μM DIDS. Recordings at the apical portion of the cell showed smaller potassium-induced extracellular H fluxes, and removal of the end foot region further decreased the H flux, suggesting that the end foot was the major source of acidifications. These studies demonstrate that self-referencing H-selective electrodes can be used to monitor H fluxes from retinal Müller cells in bicarbonate-based solutions and confirm the presence of a sodium-coupled bicarbonate transporter, the activity of which is largely restricted to the end foot of the cell. The present study uses self-referencing H-selective electrodes for the first time to measure H fluxes from Müller (glial) cells isolated from tiger salamander retina. These studies demonstrate bicarbonate transport as a potent regulator of extracellular levels of acidity around Müller cells and point toward a need for further studies aimed at addressing how such glial cell pH regulatory mechanisms may shape neuronal signaling.

Activation of retinal glial (Müller) cells by extracellular ATP induces pronounced increases in extracellular H+ flux

“…The growing body of evidence supporting the presence of microdomains of glial cell modulation [ 1 , 2 ] suggests the possibility that glial cell-mediated changes in extracellular H+ could be highly localized and might modulate highly restricted and independent sets of synaptic connections. Support for the notion of independent calcium domains within tiger salamander Müller cells includes our own previous experiments examining the effects of intracellular calcium rises induced by high extracellular potassium [ 19 ]. When tiger salamander Müller cells were tested in the same conditions as used in the present experiments (HEPES as the extracellular pH buffer with no bicarbonate present), high extracellular potassium levels induced significant increases in intracellular calcium that did not lead to any significant alteration in extracellular H+ flux.…”

“…We confirmed the lack of contribution from voltage-dependent sodium-bicarbonate exchange by examining the effects of high extracellular potassium on H+ fluxes. Depolarization of Müller cells with high extracellular potassium is known to activate the voltage-dependent sodium bicarbonate cotransporter and produce extracellular acidifications when bicarbonate is present, but not when bicarbonate is absent [ 19 , 30 , 31 ]. In our control experiments, application of 50 mM KCl failed to alter extracellular H+ flux: in 5 cells tested, a signal of 10 ± 3 μV was observed upon application of Ringer’s solution, and a signal of 18 ± 6 μV detected after addition of 50 mM KCl (p = 0.18, N = 5).…”

“…Müller cells are known to possess a voltage-sensitive, sodium coupled bicarbonate transporter that has been shown to be capable of acidifying the extracellular fluid around isolated cells [ 30 , 37 ], and we have recently reported the characterization of this transporter using self-referencing H+-selective electrodes [ 19 ]. However, the bicarbonate transporter is unlikely to account for the acidification we detect, since the experiments reported here were all done in solutions lacking added bicarbonate and in which 1 mM HEPES was the only pH buffer present.…”

“…However, the bicarbonate transporter is unlikely to account for the acidification we detect, since the experiments reported here were all done in solutions lacking added bicarbonate and in which 1 mM HEPES was the only pH buffer present. Further, challenging Müller cells with 50 mM extracellular potassium (known to activate the voltage-dependent bicarbonate transporter and alter intracellular and extracellular levels of H+ in Müller cells [ 19 , 30 ] did not induce any significant alteration in proton flux from cells, again arguing against a contribution from this transporter. Finally, DIDS and SITS are both effective in blocking the acidifying effect of the sodium-coupled bicarbonate transporter [ 19 ], while the present study shows that the extracellular H+ fluxes reported here are blocked by 300 μM DIDS but not by a comparable concentration of SITS.…”

“…Further, challenging Müller cells with 50 mM extracellular potassium (known to activate the voltage-dependent bicarbonate transporter and alter intracellular and extracellular levels of H+ in Müller cells [ 19 , 30 ] did not induce any significant alteration in proton flux from cells, again arguing against a contribution from this transporter. Finally, DIDS and SITS are both effective in blocking the acidifying effect of the sodium-coupled bicarbonate transporter [ 19 ], while the present study shows that the extracellular H+ fluxes reported here are blocked by 300 μM DIDS but not by a comparable concentration of SITS. While we do not yet know precisely what molecular mechanism(s) or transporter(s) are responsible for the observed extracellular H+ flux, the block by DIDS suggests a possible link to an anion-coupled transport system, although caution is needed even with this simple assessment, since DIDS has been suggested to be able to inhibit ATP-activated receptors directly [ 38 ].…”