Lipofuscin-formation in cultured retinal pigment epithelial cells is related to their melanin content.

Age-related macular degeneration (AMD), the leading cause of blindness in the developed world, is accompanied by degeneration of the retinal pigment epithelial (RPE) cells. There is an inverse correlation between the melanin content of the eye and the incidence of AMD. Lipofuscin (LF)-accumulation in RPE cells accompanies the process of aging, and may also be related to AMD. This study was designed to evaluate the effect of melanin/melanosomes on the rate of LF formation in cultured rabbit and bovine RPE cells subjected to oxidative stress (40% normobaric O(2)) and daily supplementation with photoreceptor outer segments for 4 weeks. The LF content was measured at 0, 2, and 4 weeks in RPE cells from pigmented and albino rabbits, as well as in pigment-rich and pigment-poor bovine cells. Albino rabbit and pigment-poor bovine cells accumulated significantly higher amounts of LF than pigmented rabbit cells and pigment-rich bovine RPE cells after both 2 and 4 weeks of exposure. Autometallography of melanin-containing cells, without previous exposure to ammonium sulfide, showed a positive outcome, indicating either the occurrence of pre-existing iron-sulphur clusters or an extremely high intrinsic reducing capacity. These results suggest that melanin acts as an efficient antioxidant, perhaps by interacting with transition metals.

Visual transduction in dialysed detached rod outer segments from lizard retina.

1. Properties of a new preparation for studying the physiology and biochemistry of phototransduction in retinal rods are described. Whole-cell voltage clamp was used to record the generation, maintenance and light-sensitivity of dark current in rod outer segments that had been isolated from the rest of the receptor cell by detachment at the connecting cilium. 2. Detached outer segments dialysed with standard internal solution supplemented with physiological amounts of ATP (5 mM) and GTP (1 mM) developed a standing inward dark current that was the sum of three components: approximately 91% light-sensitive current, approximately 6% Na(+)-Ca2+,K+ exchange current and approximately 3% leakage current. Light-sensitive dark current (mean amplitude approximately -63 pA) was suppressed transiently by brief flashes in an intensity-dependent manner. Light responses had the same kinetics, sensitivity and intensity-response relationship as those recorded from intact rods. 3. Dialysed outer segments differed from intact rods in that intense flashes evoked saturating responses that recovered incompletely to a plateau of reduced dark current caused by incomplete inactivation of the transduction cascade. Light sensitivity was reduced for a short time following an intense flash and then recovered despite persistent reduction of dark current. This suggests that there is no fixed relationship between dark current amplitude and light sensitivity. 4. Light-sensitive dark current faded rapidly when outer segments were not supplied with nucleotides. Outer segments dialysed with solution that contained cyclic GMP, but no ATP or GTP, supported dark current at a level that increased with [cyclic GMP]. When basal phosphodiesterase (PDE) activity is inhibited, 8 microM cyclic GMP supports a dark current of approximately 70 pA. 5. Light sensitivity decreased during recordings made with solution that contained only cyclic GMP, consistent with the inhibition of G protein activation by loss of GTP. After thorough nucleoside triphosphate depletion, however, intense illumination evoked a transient increase rather than a decrease in dark current, i.e. an inverted light response. This result suggests that isomerized rhodopsin may generate a signal that causes either inhibition of basal PDE activity or release of bound cyclic GMP. 6. Sustained Na(+)-Ca2+,K+ exchange current was recorded during steady illumination when Ca2+, but not when Mg2+, was added to the dialysis solution. Exchange current increased with the amount of added Ca2+ and saturated at approximately 18 pA when the dialysis solution contained > or = 10 mM Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Dual effect of local anesthetics on the function of excitable rod outer segment disk membrane.

The effects of local anesthetics and a divalent cation, Ca2+, on the function of rhodopsin were estimated from the measurements of light-induced proton uptake. The light-induced proton uptake by rhodopsin in the rod outer segment disk membrane was enhanced at lower pH (4) but depressed at higher pHs (6 to 8) by the tertiary amine local anesthetics lidocaine, bupivacaine, tetracaine, and dibucaine. The order of local anesthetic-induced depression of the proton uptake followed that of their clinical anesthetic potencies. The depression of the proton uptake versus the concentration of the uncharged form of local anesthetic nearly describes the same curve for small and large dose of added anesthetic. Furthermore, a neutral local anesthetic, benzocaine, depressed the proton uptake at all pHs between 4 and 7. These results indicate that the depression of the proton uptake is due to the effect of only the uncharged form. It is hypothesized that the uncharged form of local anesthetics interacts hydrophobically with the rhodopsin in the disk membrane. The dual effect of local anesthetics on the proton uptake, on the other hand, suggests that the activation of the function of rhodopsin may be caused by the charged form. There was no significant change in the light-induced proton uptake by rhodopsin when 1 mM of Ca2+ was introduced into the disk membrane at varying pHs in the absence or presence of local anesthetics. This fact indicates that Ca2+ ion does not influence the diprotonating process of metarhodopsin; neither does it interfere with the local anesthetic-induced changes in the rhodopsin molecule.

[Effects of oxygenated free radicals on the electrophysiological activity of the isolated retina of the rat]. / Effets des radicaux libres oxygénés sur l'activité électrophysiologique de la rétine isolée de rat.

Particularly rich in polyunsaturated fatty acids, the retina constitutes an excellent model for testing the functional consequences of membrane lipoperoxidation. The effects could be quantified by measuring the amplitude of the electroretinogram which represents the characteristic response of the retina to its physiological stimulus (light photons). The authors report results obtained on isolated rat retina maintained in survival by perfusion. The membrane lipid peroxidation is induced by the non enzymatic catalytic system (Fe2+ + ascorbate). In the presence of such a system, the amplitude of the b-wave of the electroretinogram rapidly decreases and the survival time is notably shortened. These results are discussed with regards to the mechanism generating the electroretinogram and the specific role played by the disk membrane and plasmic membrane of the rod outer segment. Finally, the authors test the radical scavenger properties of a Ginkgo biloba extract. The results indicate that such an extract develops an antiperoxidative effect probably connected with its flavonoid composition. With this extract the survival time of the isolated retina is significantly increased and this result could be related to the retinal cell membrane disorders observed in diabetes.

Light-mediated cyclic GMP hydrolysis controls important aspects of kinetics of retinal rod voltage response.

Pulsatile injections of cyclic GMP into rod outer segments of the isolated toad retina cause transient depolarizations that are reduced in amplitude in proportion with the receptor potential by low Na+ Ringer's. This reduction in the amplitude of the cyclic GMP depolarization may be due to the direct effect of external Na+ concentration on dark current and an indirect effect resulting from the inactivation of a sodium-calcium exchange mechanism raising the intracellular Ca2+ concentration. By comparison the reduction in cyclic GMP response amplitude effected by illumination is accompanied by faster kinetics. This difference suggests that the reduced amplitude and speedier response reflect a light induced increase in phosphodiesterase (PDE) activity rather than the effects of Ca2+. Large doses of cyclic GMP can distort the kinetics of both the light response and the recovery from a depolarization caused by a pulse of cyclic GMP by similarly slowing both types of responses. This similarity in the kinetics of the cyclic GMP response and the initial hyperpolarizing phase of the receptor potential suggests that the kinetics of the initial phase of the receptor potential are controlled by light-mediated cyclic GMP hydrolysis.