Effect of intracellular potassium upon the electrogenic pump of frog retinal pigment epithelium

Summary We have studied the hyperpolarizing, electrogenic pump located on the apical membrane of the retinal pigment epithelium (RPE) in anin vitro preparation of bullfrog RPE-choroid. Changes in RPE [K⁺] _i alter the current produced by this pump. Increasing [K⁺] _o in the solution perfusing thebasal membrane increases RPE [K⁺] _i (measured with a K⁺-specific microelectrode), and also depolarizes theapical membrane. This depolarization is due to a decrease in electrogenic pump current flowing across the apical membrane resistance, since it is abolished when the pump is inhibited by apical ouabain, by cooling the tissue, or by 0mm [K⁺] _o outside the apical membrane. Removal of Cl^– from the solution perfusing the basal membrane abolishes the K⁺-evoked apical depolarization by preventing the entry of K⁺ (as KCl) into the cell. We conclude that the increase in [K⁺] _i causes the decrease in pump current. This result is consistent with the finding that [K⁺] _i is a competitive inhibitor of the Na⁺–K⁺ pump in red blood cells.It is possible that the light-evoked changes in [K⁺] _o in the distal retina could alter RPE [K⁺] _i , and thus could affect the pump from both sides of the apical membrane. Any change in pump current is likely to influence retinal function, since this pump helps to determine the composition of the photoreceptor extracellular space.     

Potassium transport across the frog retinal pigment epithelium

Summary Previous experiments indicate that the apical membrane of the frog retinal pigment epithelium contains electrogenic NaK pumps. In the pressent experiments net potassium and rubidium transport across the epithelium was measured as a function of extracellular potassium (rubidium) concentration, [K] _o ([Rb] _o ). The net rate of retina-to-choroid⁴²K(⁸⁶Rb) transport increased monotonically as [K] _o ([Rb] _o ), increased from approximately 0.2 to 5mm on both sides of the tissue or on the apical (neural retinal) side of the tissue. No further increase was observed when [K] _o ([Rb] _o ) was elevated to 10mm. Net sodium transport was also stimulated by elevating [K] _o . The net K transport was completely inhibited by 10^–4 m ouabain in the solution bathing the apical membrane. Ouabain inhibited the unidirectional K flux in the direction of net flux but had not effect on the back-flux in the choroid-to-retina direction. The magnitude of the ouabain-inhibitable⁴²K(⁸⁶Rb) flux increased with [K] _o ([Rb] _o ). These results show that the apical membrane NaK pumps play an important role in the net active transport of potassium (rubidium) across the epithelium. The [K] _o changes that modulate potassium transport coincide with the light-induced [K] _o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium.     

Potassium transport across the frog retinal pigment epithelium

Previous experiments indicate that the apical membrane of the frog retinal pigment epithelium contains electrogenic Na:K pumps. In the present experiments net potassium and rubidium transport across the epithelium was measured as a function of extracellular potassium (rubidium) concentration, [K]0 ( [Rb]0). The net rate of retina-to-choroid 42K(86Rb) transport increased monotonically as [K]0 ( [Rb]0) increased from approximately 0.2 to 5 mM on both sides of the tissue or on the apical (neural retinal) side of the tissue. No further increase was observed when [K]0 ( [Rb]0) was elevated to 10 mM. Net sodium transport was also stimulated by elevating [K]0. The net K transport was completely inhibited by 10-4 M ouabain in the solution bathing the apical membrane. Ouabain inhibited the unidirectional K flux in the direction of net flux but had no effect on the back-flux in the choroid-to-retina direction. The magnitude of the ouabain-inhibitable 42K(86Rb) flux increased with [K]0 ( [Rb]0). These results show that the apical membrane Na:K pumps play an important role in the net active transport of potassium (rubidium) across the epithelium. The [K]0 changes that modulate potassium transport coincide with the light-induced [K]0 changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium.     

Passive ionic properties of frog retinal pigment epithelium

Summary The 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 conductanceT _i of each membrane. For the apical membrane:T _K0.52,T _{HCO 3}=0.39 andT _Na=0.05, and its specific resistance was estimated to be 6000–7000 cm². From the basalT _K=0.90 and its specific resistance was estimated to be 400–1200 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⁺.     

Passive ionic properties of frog retinal pigment epithelium.

The 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--20 mV). 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 conductance Ti of each membrane. For the apical membrane: TK approximately equal to 0.52, THCO3 approximately equal to 0.39 and TNa approximately equal to 0.05, and its specific resistance was estimated to be 6000--7000 omega cm2. For the basal membrane: TK approximately equal to 0.90 and its specific resistance was estimated to be 400--1200 omega cm2. From the basal potassium voltage responses the intracellular potassium concentration was estimated at 110 mM. 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 (HCO--3, Cl--, methylsulfate, isethionate) but did distinguish between Na+ and K+.     

Effect of taurine on the isolated retinal pigment epithelium of the frog: Electrophysiologic evidence for stimulation of an apical,electrogenic Na+−K+ pump

Summary The apical surface of the retinal pigment epithelium (RPE) faces the neural retina whereas its basal surface faces the choroid. Taurine, which is necessary for normal vision, is released from the retina following light exposure and is actively transported from retina to choroid by the RPE. In these experiments, we have studied the effects of taurine on the electrical properties of the isolated RPE of the bullfrog, with a particular focus on the effects of taurine on the apical Na⁺–K⁺ pump.Acute exposure of the apical, but not basal, membrane of the RPE to taurine decreased the normally apical positive transepithelial potential (TEP). This TEP decrease was generated by a depolarization of the RPE apical membrane and did not occur when the apical bath contained sodium-free medium. With continued taurine exposure, the initial TEP decrease was sometimes followed by a recovery of the TEP toward baseline. This recovery was abolished by strophanthidin or ouabain, indicating involvement of the apical Na⁺–K⁺ pump.To further explore the effects of taurine on the Na⁺–K⁺ pump, barium was used to block apical K⁺ conductance and unmask a stimulation of the pump that is produced by increasing apical [K⁺] ₀ . Under these conditions, increasing [K⁺] ₀ hyperpolarized the apical membrane and increased TEP. Taurine reversibly doubled these responses, but did not change total epithelial resistance or the ratio of apical-to-basal membrane resistance, and ouabain abolished these responses.Collectively, these findings indicate the presence of an electrogenic Na⁺/taurine cotransport mechanism in the apical membrane of the bullfrog RPE. They also provide direct evidence that taurine produces a sodium-dependent increase in electrogenic pumping by the apical Na⁺–K⁺ pump.     

Potassium modulation of taurine transport across the frog retinal pigment epithelium

Net taurine transport across the frog retinal pigment epithelium-choroid was measured as a function of extracellular potassium concentration, [K+]o. The net rate of retina-to-choroid transport increased monotonically as [K+]o increased from 0.2 mM to 2 mM on the apical (neural retinal) side of the tissue. No further increase was observed when [k+]o was elevated to 5 mM. The [K+]o changes that modulate taurine transport approximate the light-induced [K+]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. The taurine-potassium interaction was studied by using rubidium as a substitute for potassium and measuring active rubidium transport as a function of extracellular taurine concentration. An increase in apical taurine concentration, from 0.2 mM to 2 mM, produced a threefold increase in active rubidium transport, retina to choroid. Net taurine transport can also be altered by relatively large, 55 mM, changes in [Na+]o. Apical ouabain, 10(-4) M, inhibited active taurine, rubidium, and potassium transport; in the case of taurine, this inhibition is most likely due to a decrease in the sodium electrochemical gradient. In sum, these results suggest that the apical membrane contains a taurine, sodium co-transport mechanism whose rate is modulated, indirectly, through the sodium pump. This pump has previously been shown to be electrogenic and located on the apical membrane, and its rate is modulated, indirectly, by the taurine co-transport mechanism.     

Autoregulation of the electrogenic sodium pump

The dependence of electrogenic sodium pump activity on changes in the cell volume of Helix pomatia neurons with different levels of intracellular sodium ion concentration was studied. Hypertonic solutions caused hyperpolarization of the membrane and increased membrane resistance in cells with a low sodium content (low-sodium cells; LSC). The activity of the electrogenic sodium pump in hypertonic solutions was increased compared to the activity in hypotonic solutions in LSC and decreased in cells with a high sodium content (high-sodium cells; HSC). The concentration of ouabain which led to maximal inhibition of active 22Na efflux from the neurons was 10(-4) M. Lower concentrations of ouabain (10(-8) M and lower) did not inhibit the sodium pump but stimulated it. The swelling of neurons in hypotonic solutions was accompanied by an increase in the number of binding sites for ouabain, while shrinking in hypertonic solutions led to the opposite effect--a decrease in binding sites. An increase in the number of binding sites also took place in normal isotonic potassium-free solutions compared with normal Ringer's solution. Two saturable components of ouabain binding were detectable in all solutions examined. gamma-Aminobutyric acid (GABA) and acetylcholine (ACh) increased the number of ouabain binding sites on the membrane. The results suggest that there are two opposite mechanisms by which cell volume changes can modulate the pump activity. One of them depends on the intracellular sodium ion concentration and causes pump activation in hypertonic solutions in LSC and saturation in HSC, while a second mechanism mediates the activating effect of cell swelling on the sodium pump in HSC. In addition, there may be a negative feedback between the pump activity and the number of functioning pump units in the membrane.     

Ba2+ unmasks K+ modulation of the Na+-K+ pump in the frog retinal pigment epithelium

This paper presents electrophysiological evidence that small changes in [K+]o modulate the activity of the Na+-K+ pump on the apical membrane of the frog retinal pigment epithelium (RPE). This membrane also has a large relative K+ conductance so that lowering [K+]o hyperpolarizes it and therefore increases the transepithelial potential (TEP). Ba2+, a K+ channel blocker, eliminated these normal K+-evoked responses; in their place, lowering [K+]o evoked an apical depolarization and TEP decrease that were blocked by apical ouabain or strophanthidin. These data indicate that Ba2+ blocked the major K+ conductance(s) of the RPE apical membrane and unmasked a slowing of the normally hyperpolarizing electrogenic Na+-K+ pump caused by lowering [K+]o. Evidence is also presented that [K+]o modulates the pump in the isolated RPE under physiological conditions (i.e., without Ba2+). In the intact retina, light decreases subretinal [K+]o and produces the vitreal-positive c-wave of the electroretinogram (ERG) that originates primarily in the RPE from a hyperpolarization of the apical membrane and TEP increase. When Ba2+ was present in the retinal perfusate, the apical membrane depolarized in response to light and the TEP decreased so that the ERG c-wave inverted. The retinal component of the c-wave, slow PIII, was abolished by Ba2+. The effects of Ba2+ were completely reversible. We conclude that Ba2+ unmasks a slowing of the RPE Na+-K+ pump by the light-evoked decrease in [K+]o. Such a response would reduce the amplitude of the normal ERG c-wave.     

The retinal pigment epithelium of Cr-deficient rats

The purpose of this study is to investigate the effect of Cr deficiency on the rat retina. Three-week-old Wistar Kyoto rats were divided into 2 groups. Cr-deficient rats were fed AIN-93G diet without Cr and deionized distilled water. Control rats were fed AIN-93G diet and deionized distilled water. The Cr and sugar concentrations in the whole blood and cholesterol concentration in the serum were measured. We observed the retina with an electron microscope, and counted phagocytized lamellar structures in the retinal pigment epithelium (RPE) before and after the start of light exposure on negative electron microscopic films. The whole blood Cr level of Cr-deficient rats was less than 0.2 microg/l. The blood sugar level of Cr-deficient rats was significantly higher than that of normal rats (p < 0.05). There were significantly more phagocytized lamellar structures in the RPE of Cr-deficient rats 1, 2, 7, 11 and 12 h after the start of light exposure than in that of normal rats (p < 0.05). However, no morphological abnormalities were found in the photoreceptor cells of Cr-deficient rats. Phagocytosis in the photoreceptor outer segment discs in the RPE was accelerated, but the pattern of the retinal circadian rhythm with maximum phagocytosis 2 h after exposure to light was unchanged. The Cr-deficient state may cause the membrane to degenerate, and phagocytosis of the photoreceptor outer segment discs in the RPE may be accelerated. This study provided an evidence of the nutritional importance of Cr in rat retina.