Metabolism of phosphatidylethanolamine in the frog retina.

The synthesis and the turnover of phosphatidylethanolamine in frog retinal rod outer segments and microsomes were studied by monitoring the incorporation of five radioactive precursors: 32PO4, 33PO4 [3H]glycerol, [3H]serine, and [3H]ethanolamine. 1. Labeled serine was actively incorporated into phosphatidylethanolamine. The kinetics of the labeling patterns in both microsomes and rod outer segments was consistent with formation via decarboxylation of phosphatidylserine. 2. Ethanolamine was found to be an ineffective precursor of phosphatidylethanolamine, suggesting that the major pathway for phosphatidylethanolamine synthesis in the retina is via the decarboxylation reaction. 3. An active methylation of phosphatidylethanolamine to phosphatidylcholine was observed in both retinal microsomes and rod outer segments. 4. The kinetics of labeling of phosphatidylethanolamine in the rod outer segments was different for the various isotopic precursors, and was found to depend on the relative turnover times of the precursor pools. Glycerol was the only precursor that gave a true pulse of radioactivity. 5. The specific activity of phosphatidylethanolamine derived from labeled glycerol declined exponentially, demonstrating that the labeled lipid was diffusely distributed throughout the rod outer segments. The half-life of phosphatidylethanolamine in the rod outer segments was determined to be 18 days. Comparison of this value to the turnover time of rod outer segment integral proteins revealed that rod outer segment lipid is renewed at a faster rate than protein.

Metabolism of phosphatidylinositol in the frog retina.

The synthesis and the turnover of phosphatidylinositol in frog retinal rod outer segments and microsomes were studied by following the time course of incorporation into lipids of the following radioactive precursors: [3H]glycerol, 33PO4, and [3H]inositol. 1. Although all precursors were incorporated into lipid, glycerol was the only true pulse of radioactive substrate because the precursor pools of phosphate and inositol in the retina have a slow rate of turnover. 2. A precursor-product relationship exists between retinal microsomes and rod outer segments for phosphatidylinositol synthesized from glycerol. 3. The specific activity in the rod outer segment phosphatidylinositol derived from labeled glycerol was ten times that of the other glycerolipids. Since the labeled precursor for each phospholipid class is derived from a common pool of glycerol 3-phosphate, the synthesis rate of phosphatidylinositol in the retina is much greater than that of the other phospholipids. 4. Two pools of phosphatidylinositol were identified in the rod outer segments; one turned over with a t1/2 of about 3.5 days, while the other turned over at the same rate as the other phospholipids labeled with glycerol. 5. Turnover of phosphatidylinositol in the rod outer segments after glycerol injection was followed by an increase in specific radioactivity in 1,2-diacylglycerols, consistent with the latter being a lipolytic product of phosphatidylinositol in these membranes. 6. The present studies demonstrate a unique metabolism of phosphatidylinositol in the rod outer segments compared to the other phospholipids, and it is suggested that the rapid turnover of this phospholipid may be related to membrane fusion events associated with the assembly and/or turnover of rod outer segment membranes.

Metabolism of phosphatidylcholine in the frog retina.

The biosynthesis and the turnover of phosphatidylcholine were studied in the frog retina following either (a) injection into the animal of 32PO4, 33PO4, [1,3-3H]glycerol, [2-3H]glycerol, or [methyl-3H]choline, or (b) incubation of isolated retinas in solutions containing [methyl-3H]choline. 1. Examination of the pools of lipid precursors in the retina demonstrated that the choline and phosphate pools are long-lived compared to the glycerol pool, which is metabolically very active and turns over rapidly. 2. The peak in specific activity of phosphatidylcholine synthesized from labeled glycerol occurred earlier, and was higher in the microsomal fraction than in the rod outer segments, which is consistent with synthesis of phosphatidylcholine on the microsomes of the inner segment and subsequent incorporation into the rod outer segments. 3. Autoradiography of retinas incubated in vitro with tritiated choline revealed a diffuse labeling pattern in the rod outer segments. Biochemical studies following injection of labeled glycerol showed an exponential decline in specific radioactivity of phosphatidylcholine in the rod outer segments, which is consistent with a diffuse labeling of these membranes. 4. The half-life of phosphatidylcholine in the rod outer segments synthesized from labeled glycerol was found to be 18-19 days. Based on these values, calculations were made which indicated that phosphatidylcholine in the outer segments is turning over faster than integral disc membrane proteins.

Chemistry of photoreceptor membrane preparations from squid retinas.

Photoreceptor membrane preparations were made from retinas of the squid Loligo (Doryteuthis) plei for protein and lipid analysis. Lipid analysis was also completed on a single membrane preparation from Loligo pealei. (1) The membranes contain 75 wt. % protein and 25 wt. % lipid. Neutral lipids make up 26 mol % of the total lipid, the remaining 74% being phospholipid. No glycolipids were observed. (2) Free fatty acids and cholesterol comprise 8.6 and 17 mol %, respectively of the total lipid. No other neutral lipids were found. (3) Phosphatidylethanolamine and phosphatidylcholine are the major phospholipids. Lysophosphatidylcholine, lysophosphatidylethanolamine, sphingomyelin, and phosphatidylserine are present in small quantities. Phosphatidylinositol was not detected in the membranes. (4) The levels of polyunsaturated fatty acids, principally 20:4 omega 6, 20:5 omega 3, and 22:6 omega 3 are higher in the squid membranes than in any othr vertebrate or invertebrate retina that has been examined thus far. These acids account for 58 mol % of the fatty acids in phosphatidylcholine and phosphatidylserine, 75 mol % of the free fatty acids, and nearly 90% of the fatty acids of lyso- and phosphatidylethanolamine. The results from L. plei and L. pealei were indistinguishable. (5) Rhodopsin is the major protein of the membrane preparations and has a molecular weight of 50 500 +/- 850 determined by sodium dodecyl sulfate polyacrylamide gel disc electrophoresis.

Synthesis and turnover of lipid and protein components of frog retinal rod outer segments.

The incorporation and turnover of several lipid precursors in frog retina lipid and protein were studied using biochemical and autoradiographic techniques. Lipid and protein are synthesized on the photoreceptor microsomes from where some migrate to the base of the rod outer segments (ROS) and are incorporated into basal discs. Most of the labeled protein is confined to a discrete group of discs which is displaced apically as newly synthesized unlabeled discs are added. The specific radioactivity of ROS protein is constant throughout this period, which in the present study was determined to be 39 days. Conversely, lipid, once incorporated into the basal discs, diffuses throughout the entire ROS as evidenced by an exponential decline in specific radioactivity. The half-life of ROS lipid is shorter than the renewal rate for protein, suggesting that lipid turns over faster than protein in the ROS.

Catabolism of myo-inositol to precursors utilized for de novo glycerolipid biosynthesis.

Systemic injection of [2-3H]myo-inositol into frogs resulted in the incorporation of more than half of the label into glycerolipid classes other than phosphoinositides in retinal rod outer segment membranes. Following methanolysis and differential extraction of isolated lipid classes, radioactivity was recovered primarily in the aqueous phase. After phospholipase C hydrolysis of the total membrane lipids, 97% of the radioactivity was extractable with organic solvents, and 70% of the label in lipids was in 1,2-diglycerides. These results indicate that the label was incorporated primarily into the glyceryl moiety of the membrane glycerolipids. Intraocular injection of frog eyes or in vitro incubation of frog retinas with [2-3H]myo-inositol resulted in the incorporation of radioactivity almost exclusively into phosphoinositides in rod outer segment membranes. Incubation of retinas with [U-14C]glucuronic acid did not result in the formation of labeled retinal lipids. These results suggest that myo-inositol can be catabolized systemically to precursors utilized for glycerolipid biosynthesis in the retina.

The triage decision in pulmonary edema.

The authors studied the clinical courses of 216 prospectively selected patients with cardiogenic pulmonary edema presenting to an emergency ward (EW) to identify which patients should be triaged to the intensive care unit (ICU). The first four hours were considered the EW or pre-triage phase of hospitalization. During the EW phase, 108 patients remained stable; 33 of them developed cardiopulmonary complications over the next two days. Logistic regression identified four significant independent features that distinguished these 33 patients from the remaining patients: four-hour diuresis less than 1L, history of prior pulmonary edema, T-wave abnormalities, and jugular venous distention. A model containing the four variables predicted hospital complications with a sensitivity of 81% and a specificity of 65%. In comparison, the sensitivity of physicians in admitting to the ICU patients who would go on to have complications was 70%, with a specificity of 63%. In a model containing a term for the physicians' actual triage decision, all four independent predictors remained significant, producing an overall sensitivity of 81% and an overall specificity of 69%.

Phosphoinositide metabolism in the retina: localization to horizontal cells and regulation by light and divalent cations.

Isolated retinas from Xenopus laevis incorporated greater amounts of [3H]inositol and 32Pi into phosphoinositides when incubated in light than did control retinas incubated in the dark. Inositol was primarily incorporated into phosphatidylinositol (83-86%), while phosphate labeled the polyphosphoinositides (72-79%). The incorporation of radioactive glycerol, serine, choline, or ethanolamine into retinal lipids was unaffected by light. Following incubation with [3H]inositol, the cell type involved in the light response was identified by light and electron microscope autoradiography to be the horizontal cell. These results are consistent with a classic phosphatidylinositol effect in the retina. An interesting feature of this response is that the stimulus (light) is received in the photoreceptor cell and the effect is manifest in the horizontal cell.