Roy H. Steinberg

Passive ionic properties of frog retinal pigment epithelium.

SummaryThe isolated pigment epithelium and choroid of frog was mounted in a chamber so that the apical surfaces of the epithelial cells and the choroid were exposed to separate solutions. The apical membrane of these cells was penetrated with microelectrodes and the mean apical membrane potential was −88 mV. The basal membrane potential was depolarized by the amount of the transepithelial potential (8–20mV). Changes in apical and basal cell membrane voltage were produced by changing ion concentrations on one or both sides of the tissue. Although these voltage changes were altered by shunting and changes in membrane resistance, it was possible to estimate apical and basal cell membrane and shunt resistance, and the relative ionic conductanceTi of each membrane. For the apical membrane:TK⋟0.52,THCO3∼=0.39 andTNa∼=0.05, and its specific resistance was estimated to be 6000–7000Ω cm2. From the basalTK∼=0.90 and its specific resistance was estimated to be 400–1200Ω cm2. From the basal potassium voltage responses the intracellular potassium concentration was estimated at 110mm. The shunt resistance consisted of two pathways: a paracellular one, due to the junctional complexes and another, around the edge of the tissue, due to the imperfect nature of the mechanical seal. In well-sealed tissues, the specific resistance of the shunt was about ten times the apical plus basal membrane specific resistances. This epithelium, therefore, should be considered “tight”. The shunt pathway did not distinguish between anions (HCO3−, Cl−, methylsulfate, isethionate) but did distinguish between Na+ and K+.

Active transport of ions across frog retinal pigment epithelium.

Ion fluxes across the isolated frog retinal pigment epithelium-choroid preparation were measured in the short circuited state. There was a net flux of 22Na (0·47[μEq/cm2hr]) in the choroid to retina (basal to apical) direction that was 50–60% inhibited by apical ouabain, 10−4m. No seasonal variation in this net flux was observed. In contrast, the net retina to choroid 36Cl flux did vary from season to season, being high (0·5–0·74[μEq/cm2 hr]) in the spring, and low (0·2–0·5[μEq/cm2 hr]) in the summer. The net 22Na plus 36Cl flux did not account for all the short circuit current in either the spring or summer. The net 36Cl flux was equally inhibited by acetazolamide, 10−3m, and by a 10-fold reduction in the bathing solution HCO3− concentration (27·5–2·75 mm). The 36Cl fluxes were not affected by ouabain. The inhibition of the net 22Na and 36Cl fluxes exhibited a similar pattern of changes. In both cases the unidirectional flux, in the direction of net transport, was slightly or not at all reduced, and the opposite flux was significantly increased. There was a net flux of 45Ca in the choroid to retina direction of 4–5[nm/cm2 hr] that was doubled by apical ouabain (10−4m) and not affected by a 10-fold reduction in [HCO3−]0 or acetazolamide, 10−3m.

Aspects of electrolyte transport in frog pigment epithelium

This paper presents preliminary results concerning the effects of altering the electrolyte composition of the bathing media on the potential difference, and short-circuit current, and on the resting membrane potentials of pigment epithelial cells in an in vitro preparation of bullfrog pigment epithelium-choroid. Single-sided ion concentration changes showed that the apical and basal membranes of the pigment epithelium had a high K+ permeability and a relatively low permeability to Na+ and Cl−. The apical membrane was also sensitive to changes in the external HCO3− concentration. Resting membrane potentials recorded across the apical membrane were 65–90 m V with an average of 80 m V. Intracellular recordings from pigment epithelial cells provided evidence for a parallel shunt pathway which reduced the magnitude of the observed diffusion potentials. The results of removing a given ion simultaneously from the solutions bathing both sides of the tissue suggested that Na+ and HCO3− played the major roles in the production of the short-circuit current. Ouabain, 10−5m. when added to the apical solution, markedly reduced the short-circuit current and transepithelial potential.

Survival factors in retinal degenerations

Recent experiments on the retina have examined the effectiveness of various factors (e.g. growth factors, neurotrophins and cytokines) for enhancing survival and reducing injury of retinal neurons, such as photoreceptors and ganglion cells, whose death leads to blindness in degenerative retinal diseases. It has also been shown that retinal injury stimulates intrinsic survival mechanisms that promote survival of these neurons.

Chapter 2 Retinal pigment epithelial cell contributions to the electroretinogram and electrooculogram

2. Circui t o f the E R G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 2.1. Vol tages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.2. Resis tances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Effects of light and darkness on pH outside rod photoreceptors in the cat retina.

We recorded pH in the extracellular space surrounding rod photoreceptors in the dark-adapted eye of the cat and during illumination with double-barreled H(+)-selective microelectrodes. A pH of 7.17 was recorded in the vitreous at the retinal surface of the dark-adapted eye and this became more alkaline during light adaptation. In dark adaptation, a pH close to 7.00 was recorded in a region of maximal acidity in the extracellular space surrounding rods in the outer nuclear layer (ONL). pH steeply alkalinized as the microelectrode was moved more distally towards the retinal pigment epithelium (RPE), and almost reached the pH of the arterial blood at the apical surface of the RPE. Illumination produced an intraretinal alkalinization that was largest (up to 0.2 pH units) in the ONL, maximal in amplitude at rod-saturating intensities, and that was sustained during steady background illumination. The light-evoked alkalinization was relatively slow in onset, having a time constant (1/e) of 64 sec, and took 8-12.5 min to return to the dark-adapted level after the offset of maintained illumination. These results show that acid production by cat rods is highest in the dark, reflecting a high rate of energy metabolism, and suggest that glycolysis is required to support the dark current. Illumination, by suppressing both glycolysis and respiration, alkalinizes the extracellular space surrounding rods. The substantial change in pH outside rods from dark to light could alter pH dependent properties of the interphotoreceptor matrix.

Origin and sensitivity of the light peak in the intact cat eye

1. The light peak is a large light‐induced change in the DC potential across the eye (standing potential) that reaches its maximum in 5‐13 min in mammals. The light peak of the intact cat eye was studied in order to define its cellular origin and stimulus—response characteristics. Direct‐coupled recordings were made with a vitreal electrode and also with intraretinal and intracellular micro‐electrodes. Light peaks were generally evoked with 300 sec periods of diffuse white illumination.

The electrogenic sodium pump of the frog retinal pigment epithelium.

SummaryIt was previously shown that ouabain decreases the potential difference across anin vitro preparation of bullfrog retinal pigment epithelium (RPE) when applied to the apical, but not the basal, membrane and that the net basal-to-apical Na+ transport is also inhibited by apical ouabain. This suggested the presence of a Na+−K+ pump on the apical membrane of the RPE. In the present experiments, intracellular recordings from RPE cells show that this pump is electrogenic and contributes approximately −10 mV to the apical membrane potential (VAP). Apical ouabain depolarizedVAP in two phases. The initial, fast phase was due to the removal of the direct, electrogenic component. In the first one minute of the response to ouabain,VAP depolarized at an average rate of 4.4±0.42 mV/min (n=10, mean ±sem), andVAP depolarized an average of 9.6±0.5 mV during the entire fast phase. A slow phase of membrane depolarization, due to ionic gradients running down across both membranes, continued for hours at a much slower rate, 0.4 mV/min. Using a simple diffusion model and K+-specific microelectrodes, it was possible to infer that the onset of the ouabain-induced depolarization coincided with the arrival of ouabain molecules at the apical membrane. This result must occur if ouabain affects an electrogenic pump. Other metabolic inhibitors, such as DNP and cold, also produced a fast depolarization of the apical membrane. For a decrease in temperature of ≃10°C, the average depolarization of the apical membrane was 7.1±3.4 mV (n=5) and the average decrease in transepithelial potential was 3.9±0.3 mV (n=10). These changes in potential were much larger than could be explained by the effect of temperature on anRT/F electrodiffusion factor. Cooling the tissue inhibited the same mechanism as ouabain, since prior exposure to ouabain greatly reduced the magnitude of the cold effect. Bathing the tissue in 0mm [K+] solution for 2 hr inhibited the electrogenic pump, and subsequent re-introduction of 2mm [K+] solution produced a rapid membrane hyperpolarization. We conclude that the electrogenic nature of this pump is important to retinal function, since its contribution to the apical membrane potential is likely to affect the transport of ions, metabolites, and fluid across the RPE.

Rod and cone contributions to S-potentials from the cat retina

Abstract The problem of whether the rods contribute to S-potentials was studied in the intact eye of the cat. S-potentialsfrom luminosity units (L-units) were evoked by small spots of relatively monochromatic light in dark- and light-adapted retinae. The spectral sensitivity curve for dark-adapted S-potentials had its maximum at 500 nm and the form of dark-adapted responses also suggested that rods were excited. The spectral sensitivity curve for light-adapted S-potentials had its maximum at 560 nm and response latencies even at threshold were much faster than in dark-adaptation. Individual S-potentials exhibited Purkinje shifts. It was concluded that rhodopsin rods contribute to S-potentials L-type) in the cat and that cones contribute to the same responses.

Light-evoked changes in extracellular pH in frog retina

Light-induced changes in extracellular H+ concentration (delta pH0) were studied with intraretinal H(+)-sensitive double-barreled microelectrodes in frog eyecup and isolated retina preparations. The most prominent delta pH0 were found in the inner plexiform layer, as pH increases (alkalinizations) at light onset and offset. With a small-spot stimulus (0.3 mm dia.), 30 sec in duration, the delta pH0 were relatively small (0.03 pH units), and long lasting (peak at 25-30 sec). They were enhanced by flicker (0.3 Hz). Depth profiles paralleled those of the field potentials (PNR/M-wave), the ON delta pH0 peaking 40 microns more proximal than the OFF response. The delta pH0 exhibited surround antagonism, which was blocked by tetrodotoxin (TTX), indicating an independence from action potentials. The mechanism for these pH increases in proximal retina is not yet understood. In the subretinal space diffuse retinal illumination produced a small pH increase, consistent with a presumed decrease in photoreceptor lactate production. Inhibition of carbonic anhydrase (CA) with acetazolamide or methazolamide increased both the proximal and distal retinal delta pH0, suggesting that CA is involved in buffering retinal pH.