Glucose transport across isolated bovine pigment epithelium

Glucose transport across the isolated bovine pigment epithelium was studied in vitro by mounting the tissue in a chamber and exposing the apical and basal surfaces to separate solutions. Analysis of the total unidirectional glucose fluxes, for both directions, indicates that a carrier-mediated transport system exists with a significant net flux in the choroid to retina direction. The apparent kinetic constants of carrier-mediated glucose transport are: K_m = 9·5 mm, V_max = 0·71 μmol/cm²/hr for the retina to choroid direction, and K_m = 27 mm, V_max = 2·1 μmol/cm²/hr for the choroid to retina direction. Comparison of the kinetic parameters show that only the maximum velocities were significantly different. The total unidirectional glucose fluxes were insensitive to phloridzin (0·1 mm), sodium reduction (50%) and potassium reduction (90%). Phloretin (0·1 mm) and calcium reduction (90%) were effective in reducing the total unidirectional flux significantly in both directions. These findings suggest that glucose transport across bovine pigment epithelium involves a carrier-mediated transport system, and calcium is an effective modulator of the unidirectional glucose flux.     

Immunohistochemical localization of Na+/Ca2+ exchanger in human retina and retinal pigment epithelium

· Background: Impaired calcium (Ca²⁺) metabolism has been implicated in the pathogenesis of various ocular diseases, suggesting that regulation of Ca²⁺ homeostasis in the retina is of major significance for normal function. There are known families of transport proteins that can catalyze net Ca²⁺ efflux across the plasma membrane, one of which is the Na⁺/Ca²⁺ exchanger. Using immunohistochemistry, we have investigated the human retina and retinal pigment epithelium (RPE) for the presence and distribution of the so-called cardiac-type Na⁺/Ca²⁺ exchanger. · Methods: Paraffin sections of ten eyes enucleated for various disease processes were incubated according to the ABC method with a polyclonal antibody (π) produced against the canine cardiac sarcolemmal Na⁺/Ca²⁺ exchanger. The reaction was visualized with aminoethylcarbazole. · Results: There was a positive reaction with anti-Na⁺/Ca²⁺ exchanger in the retina and RPE in all eyes, but the labeling varied among the different specimens. In neural retina, staining was most intense in Mueller cells, in cells of the inner nuclear layer, and in cone inner segments. Immunoreactivity was less pronounced in ganglion cells, nerve fibers, the outer nuclear layer and in rod inner segments. Outer segments appeared mostly negative. In the RPE, positive staining was present but the intensity of staining varied both within and among the specimens. Reactive RPE cells revealed the most intense labeling. · Conclusion: Na⁺/Ca²⁺ exchanger of the cardiac type is present in human retina and RPE. The variation in immunoreactivity among the different specimens may reflect the different diseases of these eyes and their different metabolic states. A specific relation between certain diseases and malfunction of the Na⁺/Ca²⁺ exchanger could have a major impact on therapeutic regimens. Received: 19 December 1997 Revised version received: 25 February 1998 Accepted: 4 March 1998     

Relationship between short-circuit current and unidirectional fluxes of Na and Cl across the ciliary epithelium of the toad: demonstration of active Cl transport.

The unidirectional fluxes of Na⁺ and Cl^? across the isolated toad ciliary epithelium were measured under short-circuit conditions. In a normal medium, the transepithelial potential difference was about 4 mV, the epithelial side being negative with respect to the stroma side. The short-circuit current (SCC) was 46 μA/cm² and the average electrical conductance was 11 mmhos/cm². Both the Na⁺ and Cl^? fluxes from the stromal to epithelial side were greater than those in the opposite direction and the net Cl^? flux was 4 to 5 times greater than the net Na⁺ flux. In the stromal tirection, both the Na⁺ and Cl^? fluxes were linearly related to the SCC, and the sum of the partial ionic conductances of Na⁺ and Cl^? exceeded the electrical conductance. No correlation between the SCC and the Cl^? or Na⁺ flux was found in the opposite direction and the sum of the partial ionic conductance agreed well with the electrical conductance. In the presence of either theophylline in 10 mm or ouabain in 1 mm, the SCC was decreased by about 70% and unidirectional fluxes of either direction were increased in accordance with the increase in the electrical conductance, except for the stromal to epithelial Cl^? and Na⁺ fluxes in the presence of theophylline and ouabain, respectively. The data indicate that electrogenic Cl^? transport is the major function and the major source of the SCC in the epithelium.     

The influence of ions, ouabain, propranolol and amiloride on the transepithelial potential and resistance of rabbit cornea.

By means of in vitro ion substitution experiments the potential difference and resistance dependency of rabbit corneal epithelium on Na⁺, K⁺ and Cl^? activities have been established at the tear side as well as at the aqueous side. In normal conditions only a K⁺ selectivity at the aqueous side of the epithelium exists and a Na⁺ and Cl^? selectivity at the tear side. The tear side was also K⁺ selective at K⁺ activities greater than 28 mm. In Cl^? Ringer bathing solutions a rectifying current-voltage relationship is obtained. Ouabain gives rise to a two phase decrease in potential from 29·9±2·4 to 22·9±2·1 to 14·4±1·7 mV (n = 9). In the first phase the resistance changes from 3·7±0·4 to 4·0±0·4 kΩcm² (n = 9) and in the second phase R decreased to 1·3±0·1 kΩcm² (n = 9). After ouabain the Na⁺ selectivity has disappeared and the Cl^? permeability has increased. The current-voltage relationship after ouabain has become linear. Amiloride induced a reversible change in resistance from 3·5±0·6 to 4·8±0·8 kΩcm² accompanied by a depolarization from 29·1±2·7 to 22·6±1·5 mV (n = 4), moreover the rectifying properties as exhibited in control cornea are not affected by amiloride. A voltage dependent Cl^?-conductivity of the tear side membranes of the corneal epithelium can be postulated as well as a voltage independent Cl^?-conductivity that can be induced by ouabain. The ouabain effects are inhibited by propranolol.     

Calcium and the fast and slow components of P3 of the electroretinogram of the isolated rat retina

In Ca²⁺-free media the amplitudes of the a-wave (photoreceptor potential) and slow PIII of the electroretinogram of the isolated rat retina are considerably enhanced compared to the peaks observed in control medium (2 mM Ca²⁺). This enhancement requires glucose. In Ca²⁺-free media, the a-wave and slow PIII are relatively insensitive to changes of the Na⁺ concentration (25–145 m.M), while anoxia reduces but does not abolish these potentials. In normal Ca²⁺ media, the a-wave and slow PIII are insensitive to anoxia but are proportionally sensitive to reduction of the Na⁺ concentration. In Ca²⁺-free media. the a-wave and slow PIII are less affected by increasing the K⁺ concentration (5–50 mM) than in the presence of Ca²⁺. Removal of K⁺ leads to a reduction in the amplitude of the a-wave and an apparent suppression of slow PIII. Deprivation of glucose in the presence or absence of Ca²⁺ abolishes the potentials and they cannot be restored or inverted by changing the Na⁺ concentration gradient. The possible mechanisms underlying these effects of Ca²⁺ are discussed in terms of photoreceptor function.     

Oxygen transport in the bullfrog retina

In an isolated bullfrog retina preparation, macro- and micropolarographic techniques were used to measure retinal oxygen consumption in (1) the presence of active sodium transport, in light and darkness, and (2) conditions in which Na⁺ transport was inhibited by iso-osmotic Na⁺ replacement or ouabain. Total retinal consumption was measured as 4·2±0·1×10^?11 mol O₂/cm² retina/sec. Light intensities, which saturate the rod system, reduced total retinal oxygen consumption by 57%. Removal of extracellular sodium results in a decrease of 83%; similarly introduction of 10^?4m-ouabain decreases it by 86%. The greater decrease in oxygen consumption following sodium removal or ouabain was accounted for by sodium dependent oxygen utilization by cells other than the photoreceptors. The major oxygen consumption of the retina was demonstrated to occur at the level of the photoreceptor inner segments; this high oxygen consumption being necessary to maintain the high levels of active sodium transport at the photoreceptor inner segment and the concomitant interstitial dark current. The present study showing the high oxygen consumption by the photoreceptor's active sodium transport system demonstrates that the photoreceptor is important in modulating the oxygen transport from the choroidal blood to the inner retina.     

ATPase activities in retinal pigment epithelium and choroid.

The Mg^2?-, the Na⁺-K⁺- and the HCO₃^?-dependent ATPase activities of retinal pigment epithelium-choroid tissue from grassfrog, bullfrog and rabbit have been measured. In the frogs, Mg²⁺ ATPase activity was between 1 and 2 μmols P_i liberated hr^?1 mg^?1 protein, while Na⁺-K⁺ ATPase and HCO₃^? ATPase were about 20 and 25% of this activity, respectively. Corresponding values for the rabbit were 8·5 μmol P_i hr^?1 mg^?1 protein, 8% and 9%. Na⁺-K⁺ ATPase activity in each species was completely inhibited by 2 mm-calcium, while HCO₃^? ATPase was unaffected by calcium or by ouabain.The pigment epithelium-choroid preparations of bullfrog and rabbit were scraped to yield suspensions of isolated epithelial cells, relatively free of other tissue components, and a residual fraction which contained some epithelial cells, choroidal elements and red blood cells. Na^?-K⁺ ATPase activity was present in the epithelial cells of both species and absent from the choroid of the rabbit. HCO₃^? ATPase and Mg²⁺ ATPase activities were roughly equally distributed in the bullfrog fractions, but in the rabbit were concentrated in the choroid. The results are discussed in relation to the postulated active ion transport processes of the pigment epithelium.     

Homeostasis of the retinal micro-environment: I. Magnesium,potassium and calcium distributions in the avian eye

The concentrations of Mg²⁺, K⁺ and Ca²⁺ in the intraocular fluids (IOFs) and blood plasma of chickens and pigeons were determined by atomic absorption spectrophotometry. The Mg²⁺ concentration in the IOFs of both species was greatest in the liquid vitreous adjacent to the retina followed by aqueous > blood plasma > plasma dialysate. In contrast the concentration of K⁺ in the IOFs of both chickens and pigeons was greater in the aqueous than in the liquid vitreous. The concentrations of Ca²⁺ in all IOF compartments of chicken eyes were virtually identical and were lower than that of blood plasma. The concentrations of Mg²⁺ in the IOFs of the chicken, especially in the liquid vitreous, was remarkably stable; experimentally lowering or raising the plasma Mg²⁺ concentration over a relatively wide range had little or no effect on the Mg²⁺ concentration in the IOFs of these animals. We can conclude that a high Mg²⁺ and low K⁺ concentration in the extracellular fluids of the retina is maintained in the avian eye, as in the mammalian eye, by active transport processes across the blood-retinal barrier systems. Because the avian retina is completely avascular, the site of these homeostatic transport processes must be the epithelium of the retinal choroid and/or the pecten. These findings support the concept that the contribution of the vitreous to the homeostasis of the retinal micro-environment is inversely related in vertebrates to the degree of retinal vascularization.     

Thyrotropin releasing hormone: apparent receptor binding in retina.

Thyrotropin releasing hormone (TRH) binds with high affinity to sites on sheep retinal membranes. These sites closely resemble sheep pituitary receptors for TRH, as determined by direct comparison of binding in the two tissues. Specific [³H]TRH binding in sheep pituitary and retina was measured relative to blanks containing 1 μm [3-Me-His²]TRH, an analog more selective and 10-fold more potent than TRH. Incubations were at 0°C; bound radioactivity was separated by filtration. High affinity retinal TRH binding sites resembled pituitary receptors in (1) equilibrium dissociation constant (about 20–40 nm), (2) rate constant for association (about 1–2 μm^?1 min^?1), (3) rate constant for dissociation (about 0·07 min^?1), (4) effectiveness of 16 TRH analogs, ranging over six orders of magnitude in potency, in competing for binding. Major differences were (1) the retinal binding sites were present in only one quarter the concentration of pituitary receptors (about 5 pmol/g wet weight vs. 20 pmol/g wet weight) and (2) the pharmacological properties of retinal binding sites were partially obscured by the simultaneous presence of low affinity binding sites, absent in pituitary, with which 6 analogs appeared to compete more potently than for high affinity sites. These findings support the possibility that TRH or a closely related substance acts as a peptide neurotransmitter or neuromodulator in the retina.     

Histological protection by cilnidipine, a dual L/N-type Ca channel blocker, against neurotoxicity induced by ischemia-reperfusion in rat retina

Although a blockade or lack of N-type Ca²⁺ channels has been reported to suppress neuronal injury induced by ischemia-reperfusion in several animal models, information is still limited regarding the neuroprotective effects of a dual L/N-type Ca²⁺ channel blocker, cilnidipine. We histologically examined the effects of cilnidipine on neuronal injury induced by ischemia-reperfusion, intravitreous N-methyl-d-aspartate (NMDA) (200 nmol/eye) and intravitreous NOC12 (400 nmol/eye), an nitric oxide donor, in the rat retina, and compared its effects with those of ω-conotoxin MV IIA, an N-type Ca²⁺ channel blocker and amlodipine, an L-type Ca²⁺ channel blocker. Morphometric evaluation at 7 days after ischemia-reperfusion showed that treatment with cilnidipine (100 μg/kg, i.v. or 0.5 pmol/eye, intravitreous injection) prior to ischemia dramatically reduced the retinal damage. Treatment with ω-conotoxin MV IIA before ischemia (0.1 pmol/eye, intravitreous injection) significantly reduced the retinal damage. However, amlodipine (30-100 μg/kg, i.v. or 0.1-1 pmol/eye, intravitreous injection) did not show any protective effects. Treatment with cilnidipine (100 μg/kg, i.v.) reduced the retinal damage induced by intravitreous NMDA, but not NOC12. These results suggest that cilnidipine reduces Ca²⁺ influx via N-type Ca²⁺ channels after NMDA receptors activation and then protects neurons against ischemia-reperfusion injury in the rat retina in vivo. Cilnidipine may be useful as a therapeutic drug against retinal diseases which cause neuronal cell death, such as glaucoma and central retinal vessel occlusion.     

Na+-diffusion in the retinal tissue of the frog

When the isolated retina is perfused with Tyrode solution the sodium content of which has been partly replaced by sucrose the response to light shows a rapid decline to low and constant values, a process which is reversible on returning to the standard solution. The rapid drop in potential reflects the kinetics of the sodium exchange between the retina and a surrounding medium which we measured quantitatively by two methods. We first observed the change in activity of the retina preloaded with²²Na⁺ and bathed thereafter with an inactive solution. We then investigated the transretinal flux of²²Na⁺ through the retina mounted as a membrane between two chambers each of which was perfused separately. During the experiments the electroretinogram (ERG) was recorded at intervals of 5 min. From the results we calculated the diffusion coefficient of sodium which is4.47 × 10^?7 and5.35 × 10^?7 cm²/sec respectively. These values show that diffusion of sodium in the retinal tissue is greatly reduced compared to that in free solution.     

Effects of low [Ca2+]0 upon [K+]0 during and after maintained illumination of the isolated retina of the toad

Using K^?-selective microelectrodes, [K⁺]₀ was measured in the receptor layer of the isolated retina of the toad, Bufo marinus, during and after maintained adapting illumination. Lowering [Ca^2?]₀ to one-tenth of its control value produced larger, more rapid changes in [K^?]₀ at light onset and offset than under control conditions. Lowering [Ca²⁺]₀ also produced rod membrane depolarization in the dark and larger light-evoked changes in rod membrane voltage. The observed effects are consistent with a mechanism by which lowering [Ca^2?]₀ increases sodium conductance in rods in the dark, which in turn increases [Na⁺], and stimulates the rods Na⁺/K⁺ pump. This putative mechanism may be used to explain several effects of low [Ca²⁺]₀ upon rod function observed previously.     

Characteristics of ion transport across the isolated ciliary epithelium of the toad as studied by electrical measurements

Potential difference (p.d.), short-circuit current (SCC) and electrical resistance (R_t) across the isolated toad ciliary epithelium were measured under normal and various experimental conditions including altered ionic composition of the incubation medium, anoxia, the presence of 2,4-dinitrophenol (DNP), ouabain, thiocyanate (SCN^?) or theophylline. Under normal conditions, the average values of p.d., SCC and R_t were 3·7 mV (epithelial side negative), 51 μA/cm², and 88·5 Ωcm², respectively (n = 29). In ion substitution experiments, SCC increased in proportion to the increase both in Na⁺ and Cl^? concentration and showed a saturable tendency at higher concentration ranges. The elimination of either Na⁺ or Cl^? from both sides abolished p.d. and SCC almost completely. An increase in K⁺ had a stimulatory effect on SCC at lower concentrations, while it showed an inhibitory effect at higher concentrations and K⁺ (Na⁺-free)-Ringer reversed the polarity of p.d. Anoxia (bubbling with N₂), 1 mm-DNP and 1 mm-ouabain decreased p.d. and SCC when N₂ or one of the drugs was applied to either side but the effects were more potent when the drug was applied to the epithelial side. On the other hand 10 mm-SCN^? and 10 mm-theophylline caused a significant and reversible decrease in SCC only when added to the epithelial side. It is concluded that electrogenic Cl^? transport which absolutely requires both Na⁺ and K⁺ is indispensable in the transepithelial ion transport in the toad ciliary body.     

Effects of maintained illumination upon [K+]0 in the subretinal space of the frog retina

Changes in extracellular potassium ion concentration, [K⁺]₀, were measured in the subretinal space of an in vitro preparation of bullfrog retina, pigment epithelium and choroid. In response to maintained illumination, [K⁺]₀ fell to a minimum value in 30–40 sec, and then began to recover, reaching a steady-state approximately 10 min after light onset. At light offset, [K⁺]₀ overshot the dark-adapted baseline before recovering. Pigment epithelial membrane potentials followed [K⁺]₀ during this entire time period, causing parallel changes in the d.c. level of the vitreal electroretinogram (ERG). These changes in [K⁺]₀ are very similar to those observed in the cat retina in vivo, except that they are slower in time-course by nearly an order of magnitude. The vitreal ERG, however, is much different in frog than in cat, since in frog, but not in cat, it is an indirect measure of [K⁺]₀ in the subretinal space.     

Quantitative assay of phagocytosis by retinal pigment epithelium: an organ culture model

The purpose of the study was to develop a model for quantitative analysis of phagocytosis by retinal pigment epithelium (RPE). The posterior segment of the bovine eye, devoid of the neural retina and vitreous humour, formed a hollowed cup. RPE lining the interior of the cup (RPE cup) was incubated as an organ culture at 37°C for 24 hr, with latex particles (1·09 μm) previously coated with ¹²⁵I-labelled γ-globulin and then thoroughly washed. The amount of radioactivity per RPE cup was related to the phagocytic activity of the RPE cells. Electron microscopy verified that the particles were indeed internalized by RPE. Phagocytosis was significantly inhibited (P < 0·005) by iodoacetate (10^?3m), colchicine (10^?4m) and cytochalasin B (10 μg/ml). Dibutyryl c′AMP (10^?3m) and bromo-c′GMP (10^?3m) did not have any significant effect on phagocytosis.     

The effect of sodium, potassium and calcium on Limulus lateral eye retinular cells

Although the magnitude of the Limulus lateral eye receptor potential was affected by all the test solutions used, the effects on the three components were not the same. Sodium free ASW abolished C₂ and depressed C₃ more than C₁; K⁺ rich ASW depressed C₃ more than C₁ and C₂; Ca²⁺ free ASW enhanced the magnitude of C₃ significantly but had little effect on C₁ and C₂; Ca²⁺ rich ASW reduced the magnitude of all components. The reversal potential of the receptor potential was significantly reduced in Na⁺ free and Na⁺ and Ca²⁺ free ASW. Retinular cells were depolarized between 10 and 20 mV by Na⁺ and K⁺ deficient solutions and to a greater extent by K⁺ rich ASW. The effective input resistance of retinular cells always decreased in Ca²⁺ free ASW and increased in Ca²⁺ rich ASW; KCl rich solutions decreased membrane resistance and KCl deficient ASW decreased the resistance to hyperpolarizing currents but increased the resistance to depolarizing current. Na⁺ free ASW increased the effective membrane resistance except for tris-HCl substituted ASW, which decreased it. The latent period was slightly but consistently lengthened in Ca²⁺ free ASW and shortened in Ca²⁺ rich ASW. These results are considered to support the concept that the influx of Na⁺ is chiefly responsible for the production of the receptor potential and that membrane mechanisms responsible for the production of the three components of the receptor potential are different.     

Effects of variations in the perfusate on the ERG and discharge of ganglion cells in carp retina.

In order to determine the optimal condition of perfusion for maintaining a steady physiological state in the isolated carp retina, the biophysical and biochemical parameters of the perfusate were altered, and their reversible effects were investigated on the ERG and spike discharges of identified ganglion cells. A balanced salt solution for perfusion was made as the standard. When the isolated retina was perfused with the standard solution, keeping the pH at 8·0 by saturating with a gas mixture of 97% O₂ and 3% CO₂, the flow rate at 2·0 ml/min and the temperature at 20°C, a constant ERG was maintained for more than 6 hr. As the flow rate was increased from 0·6 to 13·5 ml/min, the amplitude of the b-wave was enlarged more than two times and the discharge frequency of ganglion cell also was increased. The amplitude of the b-wave was linearly related to temperature changes in the range of 14–28°C, and the Q₁₀ was 2–3. The spike discharge also increased until the temperature reached 25°C, beyond which the frequency decreased in most units. The changes in osmotic pressure between ?30 mosmol (hypotonic) and +20 mosmol (hypertonic) in comparison with the standard solution did not cause any alterations in the retinal activities. An increase of more than 30 mosmol in osmotic pressure suppressed the retinal activities; the amplitude of the b-wave was markedly reduced and the spike discharges were abolished within 40 sec of exposing the retina to 100 mosmol-hypertonic solution. pH changes between 7·0 and 9·0 were achieved either by saturating the perfusate with gas mixtures containing 0, 3, 5 and 10% CO₂ in O₂, respectively, or by titration of HCl or NaOH. A pH shift to the alkaline side (8·0–9·0) enhanced the ERG and facilitated the spike discharge, and the opposite shift (8·0–7·0) suppressed the ERG and abolished the spike discharge. The effects of pH changes on the retinal activities were found to be the same regardless of the presence of CO₂ in the perfusate. The deprivation of O₂, glucose or both of them from the perfusate gradually reduced the ERG (especially the b-wave) and the spike discharge of ganglion cells. Phosphate-rich perfusate initially activated and subsequently suppressed the spike discharge; during this period the amplitude of the a-wave was remarkably enhanced whereas the b-wave was reduced and abolished.     

The Saturation Characteristics of Glucose Transport in Bovine Retinal Pigment Epithelium

Purpose: To study the saturation characteristics of the glucose transport across the bovine retinal pigment epithelium(RPE).Methods :The bovine RPE preparations were munted with a modified Ussing chamber. The L-[3H]-glucose and 3-0-methyl-D-[14C]-glucose fluxes across the RPE from the choroid to retina were studied at different glucose concentrations.Results: The glucose transport was found to be stereospecific, with 3-0-methyl-D-glucose (MDG) being transported about three times faster than L-glucose. The glucose transport showed typical saturation characteristics in Michaelis-Menten fashion. The Vmax and the Km of corrected MDG were 2452 nmol cm-2h-1 and 30.8 mM respectively. It was shown that the glucose transport system was saturated at 61.6 mM.Conclusions : The saturation characteristics of the corrected MDG flux suggested that the capacity of glucose transport through the bovine RPE is immense. Eye Science 1998; 14: 726 -729.     

Valinomycin cataract: The relative role of calcium and sodium accumulation

Evidence was obtained which suggests that valinomycin-induced cataracts in cultured rabbit lenses are the result of calcium accumulation. Lenses exposed to 10^?5m-valinomycin for 20 hr develop superficial cortical opacities accompanied by an increase in lens Na⁺, a decrease in lens K⁺, and an increase in lens Ca²⁺. Measurable hydration is not observed. Lenses exposed to valinomycin in the presence of low concentrations of Ca²⁺ in the media do not accumulate excessive calcium and do not become opaque in spite of significant increase in lens sodium. On the other hand, lenses cultured in the presence of ouabain accumulate excessive amounts of Na⁺, lose K⁺, maintain normal Ca²⁺ levels and remain transparent. Thus, Ca²⁺ accumulation, rather than an imbalance in Na⁺ and K⁺, appears to be responsible for lens opacification.