pH Changes in Frog Rods upon Manipulation of Putative pH-regulating Transport Mechanisms

Abstract

Rod intracellular pH (pHi) in the intact frog retina was measured fluorometrically with the dye 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein under treatments chosen to affect putative pH-regulating transport mechanisms in the plasma membrane. The purpose was to relate possible pHi changes to previously reported effects on photoresponses. In nominally bicarbonate-free Ringer, application of amiloride (1 mM) or substitution of 95 mM external Na+ by K+ or choline triggered monotonic but reversible acidifications, consistent with inhibition of Na+/H+ exchange. Bicarbonate-dependent mechanisms were characterized as follows: (1) Replacing half of a 12 mM phosphate buffer by bicarbonate caused a sustained rise of pHi. (2) Subsequent application of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2',2'-disulphonic acid (DIDS, 0.2 mM) set off a slow acidification. (3) Substitution of external Cl- by gluconate (95 mM) caused a rapid pHi rise both in normal Na+ and low-Na+ perfusion. (4) This effect was inhibited by DIDS. The results support a consistent explanation of parallel electrophysiological experiments on the assumption that intracellular acidifications reduce and alkalinizations (in a certain range) augment photoresponses. It is concluded that both Na+/H+ exchange and bicarbonate transport control rod pHi, modulating the light-sensitive current. Part of the bicarbonate transport is by Na(+)-independent HCO3-/Cl- exchange, but a further Na(+)-coupled bicarbonate import mechanism is implicated.

Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic Activity

“…Dependence of Circulating Current on pH-Previously, it was proposed that the bicarbonate-induced increase in circulating current arises from intracellular alkalinization (4,27). Here we show that elevating pH does not reproduce all of the physiological effects of bicarbonate on rods described above.…”

Regulation of intracellular pH in salamander retinal rods.

“…Disregarding the possibility of a drastic species difference, there are two factors of apparent relevance to pHi. First, the total bleach in the experiments of Kalamkarov et al (1996) could have caused a large displacement of pHi at the outset, which might in turn have affected other mechanisms maintaining ion gradients, etc. Also, the rods may have been permanently hyperpolarized.…”

Regulation and Modulation of pH in the Brain

“…Ϫ exchange was found in both frog and salamander rods, as the withdrawal of Cl Ϫ produced a rise in pH i , which persisted in the absence of external Na ϩ in the case of frog rods (155). A study of retinal horizontal neurons from the skate also demonstrated the presence of amiloride-sensitive Na ϩ /H ϩ exchange, and apparent HCO 3…”

“…Early evidence for acid extrusion via Na ϩ /H ϩ exchange was provided by Oakley and colleagues (160,231) in studies of rod photoreceptors from toad. A characterization of pH i regulatory mechanisms was later carried out in rods from frog (155) and salamander retina (264). Both amphibian rods utilized an amiloride-sensitive Na ϩ /H ϩ exchanger.…”