Effects of bicarbonate versus HEPES buffering on measured properties of neurons in the salamander retina

Abstract

Electrophysiological studies of the isolated retina involve perfusing the tissue with a physiological Ringer's. Organic pH buffers such as HEPES have become increasingly popular in recent years because for many purposes they offer a convenient and reliable alternative to the more traditional bicarbonate/CO2. In this paper, however, we report that important functional properties of rods, bipolar cells, and horizontal cells in the salamander, Ambystoma tigrinum, are sensitive to the choice of buffer and, in the case of horizontal cells, that sensitivity is acute. In bicarbonate/CO2 Ringer's, the dark potential of the horizontal cell was typically near -50 mV and saturating light caused it to hyperpolarize to about -75 mV. On switching to HEPES-buffered Ringer's at the same pH, horizontal cells depolarized in darkness to about -20 mV, close to the chloride equilibrium potential, and the kinetics of their light responses changed. The cone-driven components of light responses increased in size relative to rod-driven components. Saturating lights still hyperpolarized the cells to -75 mV, however. Horizontal cells, being coupled via gap junctions, form a syncytium and syncytial length constants, measured in bicarbonate/CO2 Ringer's, were generally in the range 150-225 microm. On switching to HEPES-buffered Ringer's, length constants increased substantially to 250-330 microm. All these changes were reversible. We discuss our findings within the context of the cell's ability to regulate its internal pH.

Effects of pH Buffering on Horizontal and Ganglion Cell Light Responses in Primate Retina: Evidence for the Proton Hypothesis of Surround Formation

“…HEPES increased the light response amplitude and receptive field size of horizontal cells, consistent with a previous study (Hare and Owen, 1998) in which the normal bicarbonate buffer was replaced with HEPES. It was hypothesized that, because HEPES cannot cross cell membranes, unlike bicarbonate, intracellular pH regulation was perturbed causing the observed changes in retinal physiology.…”

Proton-Mediated Feedback Inhibition of Presynaptic Calcium Channels at the Cone Photoreceptor Synapse

“…In these cells, rollback returned (5 Ϯ 2 and 8 Ϯ 2% of peak hyperpolarizing response) after HEPES (n ϭ 3) and Tris (n ϭ 5), respectively. Hare and Owen (1998) found that the amplitude of horizontal cell light responses was dramatically increased when bicarbonate buffers were replaced with HEPES (pH 7.8) and that the cells depolarized by ϳ30 mV in addition to other effects, consistent with cytoplasmic alkalinization. In contrast, we added HEPES to the bicarbonate-buffered saline, and we did not observe such large changes in the light response.…”

Intracellular pH modulates inner segment calcium homeostasis in vertebrate photoreceptors

“…In the present study, the main mechanism regulating cytosolic acid loads under nominally bicarbonatefree conditions was the ubiquitous electroneutral Na ϩ /H ϩ antiporter, which couples proton transport to the sodium gradient (34,59). Vertebrate photoreceptors may also express electroneutral Cl Ϫ /HCO 3 Ϫ exchange (28,34,38,59) and electrogenic Na ϩ -HCO 3 Ϫ cotransport and Na ϩ -driven C Ϫ /HCO 3 Ϫ exchange mechanisms (10,83), which could be functionally coupled to carbonic anhydrases within the bicarbonate transport metabolon (62). It is likely that addition of bicarbonate and activation of cytosolic and extracellular carbonic anhydrases has additional effects on photoreceptor pH i and [Ca 2ϩ ] i regulation, outer retinal feedback, and photoreceptor cell death (10,23,83).…”

“…Studies of the blood acid-base balance in ectothermic amphibians such as Ambystoma suggest that the concentration of bicarbonate in arterial blood can vary widely over the physiological temperature range of 5 to 25°C (13a, 14a). Hence, HEPES has typically been the buffer of choice in studies that characterized pH, chloride, and calcium signaling mechanisms in isolated photoreceptors (3-4, 17, 34, 37, 39 -41, 50, 55, 60, 63-66, 68 -69) and horizontal cells (67) and has also been used in investigations of synaptic transmission at photoreceptor synapses (28,37). In the present study, the main mechanism regulating cytosolic acid loads under nominally bicarbonatefree conditions was the ubiquitous electroneutral Na ϩ /H ϩ antiporter, which couples proton transport to the sodium gradient (34,59).…”