ATP regeneration in the outer segments of frog photoreceptors]

The outer segments of retinal photoreceptors of the frog Rana temporaria were found to possess the adenylate kinase and creatine phosphokinase activities. It was shown that the activities found are not due to contaminations by the fragments of inner photoreceptor segments and pigment epithelium processes which are in direct contact with outer segments. The absolute value of the ATP resynthesis rate exceeds the rate of its hydrolysis. It is concluded that ATP synthesis in the creatine phosphokinase and adenylate kinase reactions is one of the pathways of ATP regeneration in vivo.     

Calmodulin immunolocalization in outer segments of Xenopus laevis photoreceptors

Because adaptation of vertebrate photoreceptors to light is mediated by changes in the level of calcium in their outer segments (OS), proteins that bind calcium are important in phototransduction. This study has used immunofluorescence to investigate the distribution of the calcium-binding protein calmodulin within photoreceptor OS dissociated from amphibian ( Xenopus laevis) retinas. The OS of rods and cones had a streak of fluorescence to calmodulin at the ciliary axoneme. The OS of rods (but not cones) also displayed regularly spaced puncta of anti-calmodulin fluorescence along longitudinal lines coinciding with their multiple incisures. This location of calmodulin immunofluorescence closely matches the known location of microtubules within the OS of amphibian rods and cones. These findings provide evidence that calmodulin is closely associated with the microtubules of both the axonemal and incisural cytoskeletal systems in OS, and suggest that this association is important for calmodulin function in photoreceptors.     

Lipid peroxidation and lipid peroxide detected by chemiluminescence

This article emphasizes the advantages of using a luminescence spectrometer based on photon counting techniques for the detection of lipid peroxidation. An overview is presented of how chemiluminescence can be stimulated in the luminol-cytochrome c heme peptide system as an assay for lipid hydroperoxides. This method is used for finding antioxidant drugs. The specificity and advantages of the chemiluminescent method for detecting lipid hydroperoxides is reviewed.     

Glutathione depletion increases chemiluminescence emission and lipid peroxidation in the heart

Diamide, CDNB and phorone were used to deplete glutathione in retrogradely perfused rat hearts. Following glutathione depletion the spontaneous chemiluminescence increased by 70%, irrespective of the agent used. The glutathione depletion and the chemiluminescence emission were associated to an increase of malondialdehyde content in the heart, as determined by HPLC. Under these conditions the heart function was impaired and histological examination showed a coagulative myocytolysis, a pattern already described in human and experimental pathology, where a key role is attributed to a Ca2+ homeostasis impairment.     

Enhancement of chemiluminescence associated with lipid peroxidation by rhodamine dyes.

Rhodamine Zn in concentrations of 300-500 mumole/l enhances Fe(2+)-induced chemiluminescence (CL) in blood serum, liposome and lipoprotein suspensions by two orders of magnitude. Several different rhodamines were compared, chemiluminescence spectra were measured and relationships between dye concentration, medium composition and CL intensity were studied.     

Activation of chemiluminescence accompanying lipid peroxidation in monolayer liposome suspension

In the presence of rhodamine J the chemiluminescence intensity of monolayer liposomes induced by ferrous ions is increased by three orders. There is no accumulation of malonic dialdehyde. It is suggested that chemiluminescence activation is related to rhodamine J interaction with the products of lipid peroxidation whose molecules are in the excited state.     

Dark adaptation of the photoreceptors in the isolated frog retina during induced lipid peroxidation

The influence of lipid peroxidation (LPO) in isolated frog retina on dark adaptation of photoreceptors was studied. Stimulus-response functions, late receptor potential (LRP) as function of the stimulus light intensity were measured before bleach and in a steady state after dark adaptation. It was shown that accumulation of LPO products influenced dark adaptation in photoreceptors. Based on the displacements of the stimulus-response curves and experimental measurement data on the rate of LRP collapse after retina treatment with strophanthin it is concluded that the most probable mechanism of such an influence lies in a change of photoreceptor plasma membrane permeability.     

Retinal fatty acid binding protein reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments

In the present study we have investigated the effect of partially purified retinal fatty acid binding protein (FABP) against nonenzymatic lipid peroxidation stimulated by hydroperoxides derived from fatty acids on rod outer segment (ROS) membranes. Linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) were prepared from linoleic acid, arachidonic acid and docosahexaenoic acid, respectively, by means of lipoxidase. ROS membranes were peroxidized using an ascorbate-Fe(+2) experimental system. The effect on the peroxidation of ROS containing different amounts of lipid hydroperoxides (LOOH) was studied; ROS deprived of exogenously added LOOH was utilized as control. The degradative process was measured simultaneously by determining chemiluminescence and fatty acid composition of total lipids isolated from ROS. The addition of hydroperoxides to ROS produced a marked increase in light emission. This increase was hydroperoxide concentration-dependent. The highest value of activation was produced by DHP. The decrease percentage of the more polyunsaturated fatty acids (PUFAs) (20:4 n6 and 22:6 n3) was used to evaluate the fatty acid alterations observed during the process. We have compared the fatty acid composition of total lipids isolated from native ROS and peroxidized ROS that were incubated with and without hydroperoxides. The major difference in the fatty acid composition was found in the docosahexaenoic acid content, which decreased by 45.51+/-1.07% in the peroxidized group compared to native ROS; the decrease was even higher, 81.38+/-1.11%, when the lipid peroxidation was stimulated by DHP. Retinal FABP was partially purified from retinal cytosol. Afterwards, we measured its effect on the reaction of lipid peroxidation induced by LOOH. As a result, we observed a decrease of chemiluminescence (inhibition of lipid peroxidation) when adding increasing amounts (0.2 to 0.6 mg) of retinal FABP to ROS. The inhibitory effect reaches its highest value in the presence of DHP (41.81+/-10.18%). Under these conditions, bovine serum albumin (BSA) produces a smaller inhibitory effect (20.2+/-7.06%) than FABP.     

Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors

The first step in the Visual Cycle, the series of reactions that regenerate the vertebrate visual pigment rhodopsin, is the reduction of all-trans retinal to all-trans retinol, a reaction that requires NADPH. We have used the fluorescence of all-trans retinol to study this reduction in living rod photoreceptors. After the bleaching of rhodopsin, fluorescence (excitation, 360 nm; emission, 457 or 540 nm) appears in frog and wild-type mouse rod outer segments reaching a maximum in 30-60 min at room temperature. With this excitation and emission, the mitochondrial-rich ellipsoid region of the cells shows strong fluorescence as well. Fluorescence measurements at different emission wavelengths establish that the outer segment and ellipsoid signals originate from all-trans retinol and reduced pyridine nucleotides, respectively. Using outer segment fluorescence as a measure of all-trans retinol formation, we find that in frog rod photoreceptors the NADPH necessary for the reduction of all-trans retinal can be supplied by both cytoplasmic and mitochondrial metabolic pathways. Inhibition of the reduction reaction, either by retinoic acid or through suppression of metabolic activity, reduced the formation of retinol. Finally, there are no significant fluorescence changes after bleaching in the rod outer segments of Rpe65(-/-) mice, which lack 11-cis retinal.     

The relationship between chemiluminescence and lipid peroxidation in rat hepatic microsomes.

Studies were carried out to determine the relationship between NADPH- and ascorbate-initiated chemiluminescence (CL) and lipid peroxidation (LP) in rat hepatic microsomes. NADPH-initiated CL and LP become maximal 15 min after addition of NADPH to the microsomes and ascorbate-initiated CL and LP become maximal 90 to 120 min following addition of ascorbate. There are four lines of evidence to indicate that both NADPH- and ascorbate-initiated chemiluminescence are related to lipid peroxidation. (i) The time courses for the increases in CL and in LP are identical. (ii) There is a linear relationship between total (integral) or maximal CL and LP. (iii) Drug substrates which inhibit LP also inhibit CL in a quantitatively similar manner. (iv) Inhibitors of lipid peroxidation, such as Co²⁺, Mn²⁺, Hg²⁺, para-chloromercuribenzenesulfonic acid, and EDTA, also inhibit chemiluminescence. The results of these experiments indicate that chemiluminescence initiated in hepatic microsomes by either NADPH or ascorbate is directly proportional to lipid peroxidation.     

Phosphatidylinositol stimulates phosphorylation of protein components I and II in rod outer segments of frog photoreceptors

We studied the effect of phosphoinositides on the phosphorylation of endogenous proteins in the soluble fraction of the frog photoreceptor rod outer segments (ROS). Phosphatidylinositol (PI) stimulated the phosphorylation of two low molecular weight proteins, components I and II (12 and 11 kDa) which are known to be the preferential substrates of the cyclic GMP (cGMP)-dependent protein kinase in the ROS. Polyphosphoinositides (PPI) specifically inhibited the PI-dependent phosphorylation of these two components. On the other hand, PPI stimulated the phosphorylation of 38, 48 and 52 kDa proteins in the absence of PI. These data suggest that PI and PPI may function in the ROS by regulating the phosphorylation of some enzymes or regulator proteins in the transduction mechanism in the ROS.     

Rapid declines in cyclic GMP of rod outer segments of intact frog photoreceptors after illumination

Considerable disagreement has resulted from experiments designed to test whether light-induced falls in cGMP in outer segment (OS) of photoreceptors precede their light-induced electrical responses. Different studies have reported initial declines at 50 ms, at s, or not at all for physiological stimuli. Such studies have employed whole retinas, isolated rod OS, or isolated rod OS with attached inner segments and involved a variety of techniques. We developed an apparatus that illuminates intact pieces of dark-adapted frog retinas at 22 degrees C for known brief durations and then rapidly (47 ms) presses their OS surface against a copper mirror cooled by liquid helium. Freezing occurs in less than 2 ms. Cyclic GMP was then assayed in cryostat sections of the OS layer. Six illumination intensities that bleached from 90 to 9 X 10(8) rhodopsin molecules per s were delivered for durations of 0.1-2 s. Compared to dark-adapted values, progressive losses of cGMP were seen with all illumination intensities. Because a significant loss in cGMP was seen after a 100 ms exposure to our dimmest stimulus, it appears that a loss of cGMP could play a role in rod visual transduction.     

Frog rod outer segments with attached inner segment ellipsoids as an in vitro model for photoreceptors on the retina

Purified suspensions of frog rod outer segments still attached to the mitochondria-rich inner segment portion of the receptor cell (OS-IS) can be obtained in quantities (0.1 mg/retina) sufficient for chemical analysis. In oxygenated glucose-bicarbonate Ringer's medium with added Percoll, they display normal dark currents, light sensitivity, and photocurrent kinetics for several hours. Two millimolar cytoplasmic levels of ATP and GTP are maintained, fivefold higher than in isolated OS. The levels are not altered by abolition of the dark current with ouabain. Nucleoside triphosphates are more effectively buffered than in isolated OS, and their levels remain constant during changes in external calcium levels. 32Pi is incorporated into endogenous ATP and GTP pools twice as efficiently as in isolated OS, and is used in the phosphorylation of rhodopsin. OS-IS take up and release 45Ca++ by Na+-, Ca++-, and IBMX-sensitive mechanisms. Illumination causes release of 45Ca++, which confirms retinal studies by other groups using Ca++-sensitive electrodes. Thus, OS-IS suspensions model the behavior of photoreceptors still attached to the living retina. Their availability permits the simultaneous assay and correlation of electrophysiological and chemical changes occurring during excitation and adaptation.     

Detection of in vivo lipid peroxidation using the thiobarbituric acid assay for lipid hydroperoxides

Thiobarbituric acid (TBA) assays which have been modified for detection of lipid hydroperoxides appear to be useful for demonstration of in vivo lipid peroxidation. Since these methods require heating tissue membranes with the buffered TBA, there is a possibility of interference from the detection of autoxidation that occurs during heating. These studies were undertaken to investigate conditions which favor TBA color production from hydroperoxide while limiting autoxidation during the assay. An acetic acid-sodium acetate buffered (pH 3.6) TBA assay was used. Heating linoleic acid hydroperoxide with 50 microM ferric iron or under nitrogen nearly doubled color production compared to heating it with no added iron or under air. The lipid antioxidant butylated hydroxytoluene inhibited color production from fatty acid hydroperoxides. When tissue fractions, including liver and lung microsomes and lung whole membranes, were heated in the assay, color production was greater under air than under nitrogen and was much greater under oxygen. When liver microsomes from carbon tetrachloride-exposed rats were used, color was increased only when oxygen was present in the heating atmosphere. The results with tissue fractions appear to demonstrate autoxidation during color development rather than the presence of preformed hydroperoxides. Finally, it was found that color production from membrane fractions was dependent on the vitamin E content of the membranes. It appears that autoxidation during heating should be limited by heating under nitrogen and not by adding antioxidants, which inhibit color production from hydroperoxides. As the vitamin E effect demonstrates, antioxidant status must be considered, since a change in color production could result from a change in antioxidant content without the accumulation of lipid hydroperoxides.     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.