Michael O. Hall

Kinetic studies of rod outer segment binding and ingestion by cultured rat RPE cells

Retinal pigment epithelial (RPE) cells selectively phagocytize rod outer segments (ROS) by a process which may be mediated by specific cell surface receptors. We have studied the kinetics of this process using rat RPE cells grown in tissue culture. By cooling RPE cells to 17 degrees C, the binding and ingestion phases of phagocytosis can be separated. Maximum ROS binding with minimum ingestion occurs at 17 degrees C; above 17 degrees C the rate of ingestion increases markedly. Thus it is possible to measure the kinetics of ROS binding to RPE cells at 17 degrees C and of ROS ingestion at 37 degrees C. At 17 degrees C, ROS binding is saturable, both with respect to time and to ROS concentration. ROS ingestion saturates after 4 hr of incubation at 37 degrees C, after which the cells are refractory to further ROS ingestion for 1-2 hr. During this recovery period, rapid digestion of the internalized ROS takes place. Cycloheximide, when present at a concentration (2 x 10(-5) M) which inhibits protein synthesis by 92%, has no effect on ROS phagocytosis or on the recovery of ROS ingestion at 37 degrees C. This suggests that if receptors mediate the ingestion of ROS by RPE cells, they are not degraded after the ROS are internalized. Dystrophic rat (RCS-p+) RPE cells exhibit normal binding, but very limited ingestion of ROS at 37 degrees C. The rate and amount of ROS binding to these cells at 37 degrees C is comparable with that occurring to normal cells at 17 degrees C. These observations support the hypothesis that there are a limited number of receptors which are specific for ROS binding on the surface of normal and dystrophic rat RPE cells.

Superoxide dismutase of bovine and frog rod outer segments

Abstract Bovine rod outer segments (ROS) contain soluble superoxide dismutase (SOD) which from cyanide sensitivity and electrophoretic mobility appears identical to CuZn SOD of erythrocytes. Enzyme activity of ROS extracts is 200–400 times as much as remainder of retina. Frog ROS also contains a cyanide-sensitive SOD which is not due to erythrocyte contamination since the retina is avascular. SOD in ROS may inhibit free radical oxidation of polyunsaturated fatty acids. In light, high oxygen concentrations in developing retina may activate lipid peroxidation leading to retrolental fibroplasia. High concentrations of ascorbic acid in the retina may act as a protective mechanism against superoxide.

Incorporation of [3H]vitamin A into rhodopsin in light- and dark-adapted frogs

Abstract When light- or dark-adapted frogs are injected with [3H]vitamin A acetate, the early time course of incorporation of the precursor into rhodopsin is marekedly different for the two lighting conditions. Frogs injected and kept in the light incorporate [3H]vitamin A into rhodopsin within 2 hr. In contrast, frogs injected and kept in the dark show very little labeling of rhodopsin prior to 12 hr. It appears that in light-adapted frogs, two routes of incorporation of vitamin A into rhodopsin are possible: (1) a random regeneration of rhodopsin when 11-cis-retinaldehyde combines with vacant opsin molecules in the outer segment. (2) by incorporation of 11-cis-retinaldehyde into newly forming discs at the base of the outer segment. In dark-adapted frogs, only the second process appears to occur. The time course of these two processes appears to be influenced by the activity of the pigment epithelium in light and dark.

Single-dose compared with fractionated-dose radiation of the OM431 choroidal melanoma cell line

PURPOSE To compare single-dose and fractionated-dose radiotherapeutic effects on choroidal melanoma cells. METHODS We determined the effects of gamma radiation on OM431 cell survival by exposing cells to either a single 9-Gy dose or two 4.5-Gy fractionated doses at intervals of 20 minutes to eight hours. The effects of single dosing and fractionated dosing at six hours were compared at doses of 2 to 12 Gy. RESULTS Tumor cell repair was most rapid during the first two hours. Maximum repair had occurred by six hours after radiation. Cell survival curves showed doses greater than 3 Gy of single-dose gamma radiation resulted in a greater number of cells killed than did equivalent fractionated doses. CONCLUSIONS Ocular melanoma in vitro is relatively radioresistant to low-dose fractionated radiotherapy. High single-dose radiotherapy would be more effective but would also result in more damage to normal tissue unless more focused modalities of radiotherapy are used.

Studies on the Assembly of Rod Outer Segment Disc Membranes

The vertebrate photoreceptor cell outer segment is a stack of many hundreds of closely packed membranous discs, oriented at right angles to the long axis of the cell, and enclosed within the cell membrane. The photosensitive visual pigment molecules are believed to be embedded in these discs.

Effects of gamma radiation on the OM431 human ocular melanoma cell line

In order to determine the dose responsiveness to radiation of ocular melanoma, we conducted an in vitro dose-response study on a monolayer cell culture using a clonogenic assay. The effects on cell survival were determined relative to unirradiated controls. A human epithelioid ocular melanoma cell line, OM431, was maintained in tissue culture and serial dilutions of viable cells were plated in flasks, allowed to settle and attach for 48 h, and subsequently irradiated with 1-10 Gy in single fractions. After 2 weeks, the number of reproducing clones (forming colonies with greater than 32 cells or five generations) were counted. The surviving fractions of cells were plotted on a cell survival curve using the linear quadratic model. The survival curve showed a large initial shoulder followed by an exponential decline in growth. Our data suggest that the OM431 ocular melanoma cell line responds to irradiation in a manner similar to other melanoma cell lines and is relatively radioresistant especially at lower doses.

Reduction of the retinal-opsin linkage in isolated frog retinas

Abstract A method has been developed which allows the specific reduction of the retinal-opsin Schiff base linkage in the photoreceptor cell disk membrane, using the reducing agent borane dimethylamine. The reaction is carried out in the dark at pH 1·5, conditions which minimize transiminization reactions. The product of this reaction, N -retinylopsin, can be isolated from reduced rod outer segments by column chromatography in detergent solution, or by electrophoresis in sodium dodecylsulfate polyacrylamide gels. The method is applicable to retinas which have been fixed with formaldehyde, thus allowing subsequent autoradiography of radioactive tissue. One such autoradiographic experiment showed that in the frog retina, injected [ 3 H]retinol is incorporated exclusively in the outer segments of rods and cones.

The synthesis of 3'-phosphoadenosine 5'-phosphosulfate by retinae and livers of normal and vitamin A-deficient rats.

Abstract 1. 1. A high-speed supernatant fraction from rat-retinal homogenates has been shown to catalyze the synthesis of 3′-phosphoadenosine 5′-phosphosulfate from ATP and sulfate. 2. 2. The characteristics of this system have been studied and, except for an absolute requirement for Mg2+, were found to be similar to the requirements of this system in other tissues. 3. 3. p-Chloromercuribenzoate a appeared to inhibit comletely the enzyme ATP: adenylysulfate 3′-phosphotransferase (EC 2.7.1.25) while only partially inhibiting the enzyme ATP:suulfate adenylytransferase (EC 2.7.7.4). 4. 4. While vitamin A defeciency resulted in a decreased synthesis of 3′-phosphoadenosine 5′-phosphosulfate in liver supernatants, no such effect could be shown in the retina.

The renal phospholipid composition of choline-deficient rats

The base composition of the phospholipids involved in the N-methylation pathway for the biosynthesis of phosphatidyl choline was determined in normal and severely hemorrhagic rat kidneys. There was a decrease in the proportion of phosphatidyl choline and phosphatidyl ethanolamine in the renal total lipids. The significant decrease of phosphatidyl ethanolamine in the kidney phospholipids appears to implicate this phospholipid to a greater extent than phosphatidyl choline in the etiology of the hemorrhagic syndrome.

Speckle-Tracking Strain Imaging Identifies Alterations in Left Atrial Mechanics With General Anesthesia and Positive-Pressure Ventilation

OBJECTIVES The primary aim of this study was to use speckle-tracking strain imaging to evaluate the effect of general anesthesia (GA) and positive-pressure ventilation (PPV) on left atrial (LA) mechanics. The authors hypothesized that GA and PPV would be associated with a decrease in LA strain. The secondary aims were to investigate the effects of GA and PPV on traditional Doppler-derived measures of LA function and Doppler echocardiographic grade of diastolic function. DESIGN A prospective observational study. SETTING A university hospital. PARTICIPANTS Adult patients undergoing cardiac surgery. INTERVENTIONS Transthoracic echocardiography was performed at baseline and under GA with PPV. MEASUREMENTS AND MAIN RESULTS Changes in LA function associated with GA and PPV were assessed using LA speckle-tracking strain imaging. A reduction was observed in LA peak longitudinal strain (24% v 18%, p<0.001) and preatrial contraction strain (13% v 8%, p<0.001). No difference was seen in LA contraction strain or atrial ejection fraction. Indexed LA volume and Doppler diastolic indices also were reduced significantly, and 39% of patients had a change in measured diastolic grade under GA with PPV. CONCLUSIONS Speckle-tracking strain imaging of the left atrium demonstrated that GA and PPV had a significant impact on LA mechanics by decreasing strain measures of LA preload, with a lesser effect on LA contractility.