E.L. Pautler

Glucose transport in isolated mammalian pigment epithelium

Abstract The isolated retinal pigment epithelium-choroid of sheep was studied in vitro as a single membrane mounted in a Ussing chamber. The average potential of 40 tissue preparations was 6·3 mV. In all preparations the retina (apical) side was positive with respect to the choroid (basal) side. Changes in Na + , K + , or Ca 2+ concentration on the choroid side did not affect the tissue potential, whereas a ten-fold increase in K + concentration or Ca 2+ chelation by EGTA on the retina side decreased the potential. The potential was also decreased by 0·75 m m -phloridzin and 1·0 m m -dinitrophenol. Utilizing a glucose concentration gradient of 4 m m -choroid to 1 m m -retina side, the unidirectional glucose fluxes were found to be 883 nmol/cm 2 · hr choroid to retina and 222 nmol/cm 2 · hr retina to choroid. Both unidirectional fluxes were decreased by 65% Na + replacement with choline Cl. The retina to choroid flux was also decreased by 0·75 m m -phloridzin and 1·0 m m -dinitrophenol. The sodium dependence and inhibitor effects suggest that mechanisms of glucose transport other than simple diffusion are operative in sheep retinal pigment epithelium.

Transport of acidic amino acids by the bovine pigment epithelium

The regulation of acidic amino-acid transport across the retinal pigment epithelium is of particular interest since glutamate and possibly aspartate have been identified as putative neurotransmitters in the retina, at the level of the photoreceptor cell. The present study, designed to measure the rate of acidic amino-acid transport across the mammalian pigment epithelium (PE), shows that there is a net transport of both glutamate and aspartate in the retina to choroid direction (R-C), with the R-C unidirectional flux of glutamate being substantially larger than the corresponding aspartate flux. The R-C and C-R fluxes of glutamate were found to be inhibited by ouabain. Further investigations utilizing aspartate revealed that the fluxes in both directions were inhibited when ouabain was present on the retinal side of the tissue preparation. The R-C flux of glutamate was significantly reduced by lowered concentrations of Na+, K+ and Ca2+, whereas the C-R flux was diminished only by the reduced concentration Ca2+. The changes in K+ concentration which markedly altered the R-C flux of glutamate were within the range of light-induced changes of K+ which has been observed in the extracellular space of the photoreceptor cells. The transporting system appears to be relatively specific for the acidic amino acids; for aspartate was an effective competitive inhibitor of glutamate transport whereas basic (lysine) and neutral (leucine) amino acids were not. The directionality, ouabain sensitivity, ionic dependence and substrate specificity of the transmembrane fluxes tend to support the concept of active transport as a mechanism of acidic amino-acid removal from the neural retina.

Transport of 3-O-methylglucose in isolated rat retinal pigment epithelial cells

The uptake of 3-O-methylglucose in isolated rat retinal pigment epithelial cells exhibits complex kinetics as revealed by a plot of the Lineweaver-Burke transformation of the Michaelis-Menten equation. Conventional analysis of the linear portion of the double-reciprocal plot yields a Km of 7 . 69 mM and a Vmax of 1 . 95 nM/mg protein/min. If data are analyzed to take into account the existence of two carrier-mediated processes, a high affinity system (Km = 1 . 94 mM, Vmax = 0 . 012 nM/mg protein/min) and a low affinity system (Km = 640 mM, Vmax = 12 . 20 nM/mg protein/min) for 3-O-methylglucose uptake results. Three-O-methylglucose accumulation over a 10-min period was insensitive to phloridzin (0 . 1 mM), ouabain (1 mM) and dinitrophenol (1 mM), but was inhibited by mercuric chloride (1 mM). Phloretin (0 . 1 mM) increased the accumulation of both glucose and 3-O-methylglucose, which may indicate the existence of carrier-mediated efflux system. These findings demonstrate that glucose transport in rat retinal pigment epithelial cells is a complex process involving carrier mediation.

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: Km = 9·5 mm, Vmax = 0·71 μmol/cm2/hr for the retina to choroid direction, and Km = 27 mm, Vmax = 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.

The effect of calcium on the distal P III component of the frog ERG

Abstract The distal P III component of the frog electroretinogram has been previously shown to represent a massed receptor potential of the retina. This study, utilizing isolated frog retinas, has shown that the amplitude of the distal P III potential has a near linear relationship with the concentration of calcium. The amplitude is maximal for low concentrations of calcium and is significantly reduced with increasing concentrations of calcium. Although the amplitude of the distal P III potential was greatly influenced by the calcium concentration, there was very little effect noted on the temporal recovery of response following brief intensive stimulation as a function of the calcium concentration. The enhancement of the distal P III potential found in low calcium media is also reflected in an increase in the amplitude of the b-wave, which is generated by more proximal cells of the retina. However, a slight change of “gain” was noted in the relationship between the distal P III potential and the b-wave in the low calcium concentration media. The mode of action of calcium is discussed in terms of the regulation of ionic permeabilities and active transport mechanisms.

Glucose uptake by normal and dystrophic rat retinas and ciliary bodies

Abstract The accumulation of [14C] d -glucose, [14C]3-O-methyl- d -glucose, [3H]thio- d -glucose, and [3H]2-deoxy- d -glucose was investigated in retinas and ciliary bodies isolated from normal Long-Evans rats and rats afflicted with an inherited retinal dystrophy. Rats employed in this study were taken from four age groups, ranging from 15 to 60 days. The accumulation of 2-deoxy- d -glucose and d -glucose in the normal retina decreased between 15 and 60 days. In the dystrophic retina, 3-O-methyl- d -glucose, 2-deoxy- d -glucose, and d -glucose levels decreased significantly during this time period. The decrease in accumulation of d -glucose and 2-deoxy- d -glucose in the dystrophic retina occurred at a much more rapid rate than in the normal retina, the greatest decrease occurring between 15 and 27 days. Acetazolamide, phloretin, and phloridzin significantly increased the accumulation of 3-O-methyl- d -glucose in the retina. Acetazolamide and phloretin significantly decreased the accumulation of thio- d -glucose, while acetazolamide and phloridzin increased the accumulation of d -glucose and 2-deoxy- d -glucose. The accumulation of 2-deoxy- d -glucose and d -glucose decreased significantly in normal ciliary bodies, between 15 and 60 days of age. In the dystrophic ciliary bodies, 3-O-methyl- d -glucose, d -glucose, and 2-deoxy- d -glucose intracellular levels decreased during this time period. The decline in 2-deoxy- d -glucose was much more precipitous between 15 and 27 days in the dystrophic ciliary body than in the normal ciliary body. The percent of d -glucose which was phosphorylated decreased significantly in both normal and dystrophic ciliary bodies, the decrease in the dystrophic tissue occurring earlier than in the normal tissue. Phloridzin and acetazolamide significantly increased the accumulation of thio- d -glucose and d -glucose, while phloretin increased the accumulation of 3-O-methyl- d -glucose and d -glucose in the 60-day normal ciliary body.

The effect of light on the respiration of retinas of several vertebrate and invertebrate species with special emphasis on the effects of acetylcholine and gamma-aminobutyric acid on the frog retina

Abstract The excised, dark-adapted retinas of four vertebrates [bluegill fish (Lepomis macrochirus), leopard frog (Rana pipiens), white leghorn chicken (Gallus gallus), and the white laboratory rat (Rattus rattus)], and of one invertebrate (Octopus bimaculatus), were examined by electronic respirometry to test the effect of darkness and flicker light (10 flashes/sec) on oxygen uptake. In all cases but one, respiration in flicker light was lower than in darkness. The reverse was true in the octopus retina. Further experiments were performed on the frog retina which tested the effect of a number of compounds on respiration. Of those that are known to be present in retinas, 10 μ m - and 100 μ m -gamma-amino butyric acid (GABA) decreased oxygen uptake to the level normally found in flicker light, and 10 μ m - and 100 μ m -acetylcholine (ACh) increased oxygen uptake to the level normally found in darkness. When the effect of various drugs was tested on respiration, both 100 μ m -d-Tubocurarine and atropine reversed the normal pattern of respiration, with the frog retinas absorbing oxygen faster in the light than in the dark. Eserine inhibited the effect of flicker light, and 3-pyrazolidone, the cyclic form of hydrazinopropionic acid a compound with structural similarity to GABA, had the same inhibitory effect as did GABA. On the strength of these data and observations reported in the literature, we suggest a possible mechanism for the regulation of respiration in darkness and light based on control by ACh and GABA. The normal respiratory photic effect was observed in the presence of 10 m m -aspartic acid, but ACh, GABA and atropine had no effect on either the PII or PIII components of the electroretinogram. We therefore conclude that the light-activated events to be seen on the electroretinogram cannot be caused by the respiratory changes that are induced in the retina by light or darkness. Their possible relationship to the visual process is discussed.

The effect of iodoacetate on the retinal cell DNA in rabbit

Abstract Sedimentation of the DNA from control rabbit retinal cells revealed a major peak at 580 Svedberg units. Following a single injection of 20 mg/kg of sodium iodoacetate (IAA), the percent of DNA in this control peak showed a rapid decline which persisted for about 3 hr. From 5–10 hr after injection, the percent of DNA in the control peak increased after which a secondary, slower decline occurred which levelled off at about 20 hr and then persisted for at least 5 days. Electron microscopy demonstrated changes in the chromatin material of the visual cells during the period of investigation. Electroretinograms were depressed shortly after injection but were completely recovered 6 hr later and remained normal throughout the experimental period. It is suggested that IAA induces an initial damage to the DNA which is capable of being repaired but that a concomitant slower toxic action results in irreversible damage to a portion of the DNA. The residual damage to the DNA may be related to the “latent” effect of IAA toxicity observed by other investigators.