Hih-Min Wang

Adaptive changes in visual cell transduction protein levels: Effect of light

Long-term environmental light-mediated changes in visual cell transduction proteins were studied to assess the influence of rearing environment on their levels and their potential effects on intense light-induced retinal damage. The levels of rhodopsin, S-antigen and the alpha subunit of transducin were measured in whole eye detergent extracts, retinal homogenates or rod outer segments isolated from rats reared in weak cyclic light or darkness, and following a change in rearing environment. Rats changed from weak cyclic light to darkness had 22% more rhodopsin per eye than cyclic-light rats after 12-14 days in the new environment. Western trans-blot analysis of retinal proteins from these dark-maintained animals contained 65% higher levels of immunologically detectable alpha transducin; S-antigen levels were approximately 45% lower than in cyclic-light rats. In rats changed from the dark environment to weak cyclic light, rhodopsin levels decreased by 18% during a comparable period; retinal alpha transducin was 35% lower, S-antigen was 30% higher. At various times after the change in rearing environment, some rats were exposed to intense visible light to determine their susceptibility to retinal damage. Two weeks after an 8-hr exposure, cyclic-light reared rats had rhodopsin levels only 10% lower than control (2.1 nmol per eye). However, rhodopsin was 75% lower when cyclic-light rats were maintained in darkness for 2 weeks before intense light. For animals originally reared in darkness, rhodopsin was 78% lower following 8 hr of intense light, whereas only 30% rhodopsin loss occurred in dark-reared rats after previous maintenance for 2 weeks in weak cyclic-light.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascorbate and glutathione levels in the developing normal and dystrophic rat retina: effect of intense light exposure

Ascorbic acid and glutathione were measured in retinas excised from normal rats reared in a cyclic light or dark environment and in dystrophic rats from the dark environment. Similar measurements were made on retinas from age matched rats exposed to intense visible light for periods of up to 24 hours. In other rats, ascorbic acid was given for various periods before exposure to intense light and the degree of photoreceptor cell death determined subsequently by rhodopsin measurements. In non-intense light treated rats ascorbate and glutathione were 12.1 nmol/retina at 20 days of age and 13.3 - 15.9 nmol/retina in 60 day old animals. In dystrophic rat retinas glutathione was 4-8% higher and ascorbate 10-20% higher than in normal dark reared rats. Although the levels of ascorbate and glutathione per retina increased during development, the molar ratios of the antioxidant materials to rhodopsin decreased by 36% and 60% in normal and dystrophic rats respectively. The levels of glutathione in young cyclic light or dark reared normals were unaffected by intense light exposure of either short (2-4 hrs) or long (24 hrs) duration. However, in both 20 and 40 day old dystrophic rats, intense light exposure resulted in a significant increase in retinal glutathione. In contrast to glutathione, retinal ascorbate decreased in normal rats exposed to intense light for 24 hrs, in an age and prior light environment dependent fashion. At ages greater than 20 days, normal rats exposed to light had significantly lower retinal ascorbate levels than their non-light exposed counterparts. The levels of ascorbate in 21-40 and 41-60 day old dark reared rat retinas were also significantly lower than in comparable intense light treated-cyclic light reared rats. In the youngest dystrophic rats whole eye ascorbate (retina, RPE, choroid and sclera) was 20-30% lower than in non-light treated rats, but in older mutant rats (41-60 day) light had no effect on the level of ascorbate in the retina. As determined by the level of rhodopsin remaining in the eye two weeks after 24 hrs light exposure, cyclic light reared rats lost 50-55% of their visual cells. However, cyclic light rats supplemented with ascorbic acid before intense light exposure lost only 30-35% of their visual cells.

α-Tocopherol in the developing rat retina: a high pressure liquid chromatographic analysis

High pressure liquid chromatography was used to measure a-tocopherol in the retinas of rats reared in a cyclic light or dark environment. These measurements were performed on extracts of whole retinas during the developmental period, 18–60 days, and on isolated ROS from adult animals. Similar α-tocopherol determinations were performed on retinas and isolated ROS following exposure of rats to intense visible light for 24 hr periods.The results show that a-tocopherol is chromatographically separated from the vitamin A derivatives found in the retina and is pure, as judged by mass spectrometry. In the retinas of cyclic light and dark reared rats, a-tocopherol accumulates in an age dependent fashion, so that at 60 days the level is nearly double that of animals at 18–20 days of age (P<0.001). Because the age dependent accumulation of rhodopsin is greater in dark reared rats, the average molar ratio of rhodopsin to α- tocopherol in the retina of dark reared animals is 25% higher than in cyclic light rats. Foll...

The enzymatic estimation of organic hydroperoxides in the rat retina

An enzymatic procedure for the estimation of organic hydroperoxides has been adapted to biological tissues and applied to the measurement of hydroperoxides in the rat retina. Hydroperoxides are determined from the coupled activities of glutathione peroxidase and glutathione reductase as measured by the loss of NADPH absorbance. To minimize the effects of tissue catalyzed peroxide degradation, incubations were performed in the presence of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU); which inhibited the activity of retinal tissue glutathione reductase by 85%. For comparisons to the enzymatic technique, retinal tissue hydroperoxides were also estimated by the absorption of tissue extracts at 232 nm. Using the enzymatic procedure the hydroperoxide concentration in whole retina homogenates was significantly higher in 19-day-old rats than in either 35-day or adult animals. Hydroperoxides in the retina of young rats exposed to light for one hour were significantly lower than in non-exposed controls, while in adult rats, following light, hydroperoxides increased 13%. Fractionation of rat retinas into crude ROS and retina minus ROS components revealed that the ROS fractions contain at least twice the hydroperoxide concentration of the remaining retina. The concentration of hydroperoxides in the ROS fractions from dark-reared rats were significantly lower than in cyclic-light-reared animals. In both types of rats, one hour intense light exposure resulted in an increase in ROS hydroperoxides but the increases were not significant. ROS hydroperoxides were also found to be 85-90% water soluble. Estimates of the retinal hydroperoxide content obtained by absorption at 232 nm gave similar results to the enzymatic technique, but the levels were significantly lower. When retinas were maintained in vitro for one hour before analysis, hydroperoxides determined by either technique were significantly higher than in retinas assayed immediately, but A232 hydroperoxides were still significantly lower than hydroperoxides measured by the enzymatic procedure. It is concluded: (1) that the observed retinal hydroperoxide concentration depends upon animal age and the method of measurement; (2) that within the retina the photoreceptor cell contains at least a two-fold higher concentration of hydroperoxides than the remaining retina and that prior light history can affect those hydroperoxide levels (it appears that the photoreceptor cell is also a major site of hydroperoxide formation in the retina); (3) that during intense light exposure of short duration significant levels of hydroperoxides do not accumulate in the retinas of rats.

Rod outer segment lipid--opsin ratios in the developing normal and retinal dystrophic rat.

Abstract The outer segment membrane lipid and opsin contents were determined in photoreceptor cell rods isolated from the eyes of developing normal rats reared in cyclic light or dark environments and dark-reared dystrophic rats. In cyclic light-reared normals rhodopsin/eye increased 49% during the period 20–60 days. Total ROS lipid content, a measure of ROS length, increased 50% while the polyunsaturated fatty acid docosahexaenoate increased from 42–51 mol % during the same period. The phospholipid/opsin ratio of cyclic light reared rat ROS membranes was 67 mol/mol at 20 days and 68 mol/mol at 60 days. In young dark-reared normals the phospholipid/opsin ratio was the same as for cyclic light-reared rats. Although 60 day-old dark-reared normals had 30% more rhodopsin/eye than their cyclic light-reared counterparts, non-significant changes in ROS length (14% longer) and in the phospholipid/opsin ratio (8% lower) were measured in these rats. In addition, light deprivation had no significant effect on the concentrations of polyunsaturated fatty acids or the lipid composition of the isolated ROS. The eyes of dark-reared rats with retinal dystrophy accumulated two times more rhodopsin than dark-reared normals during the 20–60-day period. The phospholipid/opsin ratio of mutant rat ROS was only 7% lower than dark normal at 20 days and 13% lower at 34 days. However, by 60 days of age, the phospholipid/opsin ratio in dystrophic rat ROS was significantly lower than in ROS from either cyclic light-or dark-reared normals. Docosahexaenoic acid in mutant rat ROS lipids averaged 40 mol% during the developmental period. These levels were significantly lower than the levels of docosahexaenoate measured in dark normals at both the 34- and 64-day periods. The glycerophospholipid composition of dystrophic rat ROS was the same as normal at all ages but the cholesterol/phospholipid ratio was higher than in normals. The data show: (1) that the retina accomodates changes in rhodopsin content induced by environmental light, age and genetic differences by alterations in ROS opsin density and length: (2) that the content of ROS membrane polyunsaturated fatty acids (fluidity) increases during development in normals but not in dystrophic rats. The data also suggest that basal membrane synthesis and/or post sythetic membrane modification of ROS lipid are impaired as a function of age in dystrophic rats.

Vitamin E in human neural retina and retinal pigment epithelium: Effect of age

Vitamin E levels were measured in retina and retinal pigment epithelium from human eye bank donors of from 12-82 years of age. In comparison to an age group of 12-45 years, humans 59-82 years of age had a higher concentration of vitamin E in both retina and retinal pigment epithelium. Depending on age, the concentration of vitamin E in retinal pigment epithelium was from 4-7 times higher than in retina. Vitamin E accumulated in the human retinal pigment epithelium in an age dependent fashion, so that by 80 years it was from 3-4 times higher than in those 20 years old. The level of vitamin E in young human retinal epithelium, however, was higher than in comparable bovine tissue. The age-related increase in human tissue vitamin E levels does not appear to be affected by postmortem time.

Long term effects of diaminophenoxypentane in the rat retina: protection against light damage

The retinotoxic drug diaminophenoxypentane (DAPP) was administered to rats to determine its long term effects on rhodopsin levels, retinal morphology and the retinas susceptibility to damage from visible light. In rats given 2 intraperitoneal injections of DAPP at doses of 65 mg/kg body wt, there was a dramatic and sustained loss of rhodopsin. One wk later visual pigment levels were 41% lower than in comparable dark maintained rats injected with saline. Rhodopsin levels in the DAPP treated rats remained lower than in control animals for the 13 wk period of the study. Morphologically, the ROS of rats 1-2 wks after DAPP treatment exhibited some disorganization and shortening; the RPE was unremarkable. Seven wks after DAPP treatment an occasional focal area of damage was seen in the RPE. Similarly, focal areas of degeneration were seen in the outer nuclear layer between the rows of photoreceptor cells. As determined by photoreceptor cell nuclear counts, the retinotoxic effect of DAPP persisted long after drug administration. In the treated rats the loss of visual cell nuclei was 11% at 7 wks; it was 22% 13 wks after DAPP treatment. Immediately after exposure to intense visible light, damage was seen in both the photoreceptor cells and RPE of DAPP treated rats. However, the effects of exposure in the treated rats were less dramatic than in retinas from rats without DAPP treatment. Following a 2 wk dark recovery period, the DAPP treated rats had a normal appearing retinal morphology and an intact RPE layer. The retinas of rats without DAPP treatment showed extensive visual cell and RPE loss.(ABSTRACT TRUNCATED AT 250 WORDS)