Extracellular H(+) has been hypothesized to mediate feedback inhibition from horizontal cells onto vertebrate photoreceptors. According to this hypothesis, depolarization of horizontal cells should induce extracellular acidification adjacent to the cell membrane. Experiments testing this hypothesis have produced conflicting results. Studies examining carp and goldfish horizontal cells loaded with the pH-sensitive dye 5-hexadecanoylaminofluorescein (HAF) reported an extracellular acidification on depolarization by glutamate or potassium. However, investigations using H(+)-selective microelectrodes report an extracellular alkalinization on depolarization of skate and catfish horizontal cells. These studies differed in the species and extracellular pH buffer used and the presence or absence of cobalt. We used both techniques to examine H(+) changes from isolated catfish horizontal cells under identical experimental conditions (1 mM HEPES, no cobalt). HAF fluorescence indicated an acidification response to high extracellular potassium or glutamate. However, a clear extracellular alkalinization was found using H(+)-selective microelectrodes under the same conditions. Confocal microscopy revealed that HAF was not localized exclusively to the extracellular surface, but rather was detected throughout the intracellular compartment. A high degree of colocalization between HAF and the mitochondrion-specific dye MitoTracker was observed. When HAF fluorescence was monitored from optical sections from the center of a cell, glutamate produced an intracellular acidification. These results are consistent with a model in which depolarization allows calcium influx, followed by activation of a Ca(2+)/H(+) plasma membrane ATPase. Our results suggest that HAF is reporting intracellular pH changes and that depolarization of horizontal cells induces an extracellular alkalinization, which may relieve H(+)-mediated inhibition of photoreceptor synaptic transmission.

Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial) cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

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.

Small alterations in extracellular H+ can profoundly alter neurotransmitter release by neurons. We examined mechanisms by which extracellular ATP induces an extracellular H+ flux from Müller glial cells, which surround synaptic connections throughout the vertebrate retina. Müller glia were isolated from tiger salamander retinae and H+ fluxes examined using self-referencing H+-selective microelectrodes. Experiments were performed in 1 mM HEPES with no bicarbonate present. Replacement of extracellular sodium by choline decreased H+ efflux induced by 10 µM ATP by 75%. ATP-induced H+ efflux was also reduced by Na+/H+ exchange inhibitors. Amiloride reduced H+ efflux initiated by 10 µM ATP by 60%, while 10 µM cariporide decreased by 37%, and 25 µM zoniporide reduced H+ flux by 32%. ATP-induced H+ fluxes were not significantly altered by the K+/H+ pump blockers SCH28080 or TAK438, and replacement of all extracellular chloride with gluconate was without effect on H+ fluxes. Recordings of ATP-induced H+ efflux from cells simultaneously whole-cell voltage-clamped revealed no effect of membrane potential from -70 mV to 0 mV. Restoration of extracellular potassium after cells were bathed in 0 mM potassium produced a transient alteration in ATP-dependent H+ efflux. The transient response to extracellular potassium occurred only when extracellular sodium was present and was abolished by 1 mM ouabain, suggesting alterations in sodium gradients mediated by Na+/K ATPase activity. Our data indicate that the majority of H+ efflux elicited by extracellular ATP from isolated Müller cells is mediated by Na+/H+ exchange.

Small alterations in the level of extracellular H+ can profoundly alter neuronal activity throughout the nervous system. In this study, self-referencing H+-selective microelectrodes were used to examine extracellular H+ fluxes from individual astrocytes. Activation of astrocytes cultured from mouse hippocampus and rat cortex with extracellular ATP produced a pronounced increase in extracellular H+ flux. The ATP-elicited increase in H+ flux appeared to be independent of bicarbonate transport, as ATP increased H+ flux regardless of whether the primary extracellular pH buffer was 26 mM bicarbonate or 1 mM HEPES, and persisted when atmospheric levels of CO2 were replaced by oxygen. Adenosine failed to elicit any change in extracellular H+ fluxes, and ATP-mediated increases in H+ flux were inhibited by the P2 inhibitors suramin and PPADS suggesting direct activation of ATP receptors. Extracellular ATP also induced an intracellular rise in calcium in cultured astrocytes, and ATP-induced rises in both calcium and H+ efflux were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin. Replacement of extracellular sodium with choline did not significantly reduce the size of the ATP-induced increases in H+ flux, and the increases in H+ flux were not significantly affected by addition of EIPA, suggesting little involvement of Na+/H+ exchangers in ATP-elicited increases in H+ flux. Given the high sensitivity of voltage-sensitive calcium channels on neurons to small changes in levels of free H+, we hypothesize that the ATP-mediated extrusion of H+ from astrocytes may play a key role in regulating signaling at synapses within the nervous system.