Light- and nucleotide-dependent binding of phosphodiesterase to rod disk membranes: correlation with light-scattering changes and vesicle aggregation

Under conditions in which large guanosine cyclic 3',5'-phosphate (cGMP)- and phosphodiesterase (PDE)-dependent changes in near-infrared transmission and vesicle aggregation and disaggregation occur, we have observed a striking change in the binding of PDE to rod disk membranes. The change in PDE binding is nucleotide and light dependent as are the light-scattering changes. The cGMP- and PDE-dependent light-scattering signal can be produced by a 500-nm light flash which bleaches 1/(1 X 10(7] rhodopsin molecules. Mg ions are an essential cofactor for the nucleotide-dependent PDE binding and light-scattering changes. 3-Isobutyl-1-methylxanthine and other competitive inhibitors of PDE hydrolytic activity support increased PDE binding to the disk membrane, vesicle aggregation, and the light-scattering signal. However, treatments which block GTP-dependent activation of PDE hydrolytic activity (colchicine, GDP, or ethylenediaminetetraacetic acid) also block these phenomena. Thus, GTP-dependent activation of PDE rather than its hydrolytic activity appears to be correlated with the light-scattering signal.     

Evidence for aerobic metabolism in retinal rod outer segment disks

The disks of the vertebrate retinal rod Outer Segment (OS), devoid of mitochondria, are the site of visual transduction, a very energy demanding process. In a previous proteomic study we reported the expression of the respiratory chain complexes I–IV and the oxidative phosphorylation Complex V (F₁F₀-ATP synthase) in disks. In the present study, the functional localization of these proteins in disks was investigated by biochemical analyses, oxymetry, membrane potential measurements, and confocal laser scanning microscopy. Disk preparations, isolated by Ficoll flotation, were characterized for purity. An oxygen consumption, stimulated by NADH and Succinate and reverted by rotenone, antimycin A and KCN was measured in disks, either in coupled or uncoupled conditions. Rhodamine-123 fluorescence quenching kinetics showed the existence of a proton potential difference across the disk membranes. Citrate synthase activity was assayed and found enriched in disks with respect to ROS. ATP synthesis by disks (0.7 μmol ATP/min/mg), sensitive to the common mitochondrial ATP synthase inhibitors, would largely account for the rod ATP need in the light.Overall, data indicate that an oxidative phosphorylation occurs in rod OS, which do not contain mitochondria, thank to the presence of ectopically located mitochondrial proteins. These findings may provide important new insight into energy production in outer segments via aerobic metabolism and additional information about protein components in OS disk membranes.     

Inositol-less death in yeast results in a simultaneous increase in intracellular viscosity.

Inositol auxotrophs of yeast developing on isositol-deficient medium continue protein synthesis for 4-6 h, lose viability rapidly after 6 h, and show an increase in cytoplasmic viscosity as measured by spin label rotational motion. Cycloheximide prevents the rapid loss of cell viability, stops protein synthesis, and simultaneously prevents an increase in cytoplasmic viscosity. From these observations, we infer that intracellular translational diffusion is upset as a consequence of inositol starvation. Cell death may be caused by a modified intracellular diffusion environment.     

Effect of sonication on nucleotide-dependent light scattering changes in retinal rod outer segment suspensions.

Near-infrared light scattering from suspensions of rod outer segment fragments is a useful probe of visible-light-activated changes in peripheral membrane proteins in photoreceptor cells. Limited sonication of suspensions has been shown to increase the amplitude of light induced turbidity changes in the presence of guanosine triphosphate by a factor of 2. Further sonication led to a decrease in the signal amplitude by an order of magnitude. This reduction has been puzzling, since the activity of the GTP-binding protein (as measured by GTP hydrolysis turnover number) was unaffected by the range of sonication used. This effect of sonication is investigated here using a novel, Reticon-based apparatus that measures the angular distribution of scattered light from samples as small as 1 microliter. The results show that even at high rhodopsin concentrations (125 microM) with millimeter path lengths, significant amounts of unscattered light are transmitted by the samples. A simple phenomenological theory that assumes a constant fractional change in scattering power (15%), independent of amount of sonication, explains the effect of sonication on the angle dependence data as well as the original turbidity data. The results have general relevance for optimization of light-scattering studies of membrane systems.     

A drop in "apparent" blood viscosity due to administration of high-molecular polymer capable of augmenting viscosity

Influence of a high-molecular compound capable of augmenting viscosity, namely: polyethylene oxide Polyox WSR-301, on hemodynamic parameters in rat mesenteric microvessels was investigated. A substantial decrease in the arteriolar hemodynamic resistance caused by the polymer was revealed. Special research has shown that this reaction is not connected with a vasodilatation and, therefore, is caused by a reduction in the "apparent" viscosity of the blood, i.e., it is a consequence of changed properties of the blood flow.     

Calcium adenosinetriphosphatase of bovine retinal rod outer segment disks.

A Ca(2+)-pumping ATPase activity is present in bovine retinal rod outer segment purified disks. The ATPase has a high Ca2+ affinity (KM = 25 microM). Low Ca2+ (n-microM) concentrations stimulate an ATP-dependent Ca2+ uptake and the ATP hydrolysis in the absence of exogenous Mg2+. Electrophoretic analysis of disk proteins after treatment with (gamma-32P)ATP shows the existence of the enzyme-phosphate acid-stable, hydroxylamine-sensitive intermediate complex of molecular mass of about 135 kDa. The results would indicate the presence of an inwardly directed Ca(2+)-ATPase pump acting on the disk membrane, that could be involved in the regulation of cytosolic free Ca2+ levels inside ROS.     

Effect of cGMP and cations on the permeability of cattle retinal disks

Guanosine 3',5'-monophosphate and Na or Ca ions affect the transmembrane movements of the same pool of intradiskal ions. Extradiskal Na ions activate the efflux of intradiskal Na ions. Extradiskal Ca ions activate the efflux of intradiskal Rb ions. Na and Ca ions activate Na/Ca or Ca/Ca exchange, as previously described. cGMP activates a membrane permeability for all the cations tested, as previously described. The reciprocal relations between cGMP and the other pathways for ion movements through disk membranes are systematically examined. Some analogies between the cGMP-activated permeability of the disk membranes and the light-sensitive conductance of the rod plasma outer membrane are discussed.     

A semi-empirical model of apparent blood viscosity as a function of vessel diameter and discharge hematocrit

A semi-empirical model is developed to describe the dependence of apparent viscosity of blood on vessel diameter (2.7 to 500 microns) and vessel discharge hematocrit (5% to 60%). The blood flow is modeled as a cell-rich core and a cell-free marginal layer in the larger vessels and an axial-train in the smaller vessels. Laminar (Poiseuille) flow is assumed in all cases. An equation is derived in which apparent viscosity is a function of vessel diameter, core viscosity, and width of marginal layer. This is then complemented by empirical equations in which core viscosity varies exponentially with discharge hematocrit while the width of marginal layer varies linearly with discharge hematocrit. The model correlates well with several sets of experimental data and behaves according to the Fahraeus-Lindqvist effect. Predicted apparent viscosity tends to the expected finite value for large vessel diameters. Dependence of apparent viscosity on vessel diameter is realistically smooth in the whole diameter range.     

Elevation of apparent membrane viscosity in ovarian granulosa cells by follicle-stimulating hormone

Continued exposure of cultured granulosa cells to follicle-stimulating hormone (FSH) induced: (i) a rise in apparent membrane microviscosity, as reflected by an increase in fluorescence polarization of the lipid-soluble probe, 1,6-diphenyl-1,3,5,-hexatriene; and (ii) a progressive decline in the cyclic AMP response to renewed challenge with the same hormone. Both changes were reduced or prevented by pretreatment of the cells with oleic or linoleic acid, agents which reduce membrane viscosity, but not by elaidic or palmitic acid which increase the rigidity of membrane lipids. Other agents that inhibited FSH-induced changes in membrane fluidity (gonadotropin-releasing hormone, actinomycin D and cycloheximide) also prevented desensitization to FSH. Cyclic AMP and cyclic GMP derivatives did not mimic the effects of FSH on apparent membrane viscosity or desensitization. Changes in membrane fluidity are unlikely to be the sole cause of desensitization since (i) pretreatment of the cells with fatty acids that increase lipid viscosity did not induce desensitization to FSH, and (ii) desensitization of granulosa cells to lutropin and prostaglandin E2 by exposure to the homologous hormone was not attended by increased membrane viscosity. The experiments described provide the first example of a hormonally induced increase in the target cell apparent membrane viscosity.     

Light changes the membrane potential and ion balances of retinal rod disks

Light stimulation of rod cells in vertebrate eyes may cause Ca2+ release from the intracellular disks. Radiolabelled tracers show that light causes a small hyperpolarization of intact disk stacks and redistribution of the ions Ca2+ and Cl-.     

The apparent viscosity of aggregating and non-aggregating erythrocyte suspensions in the isolated perfused liver

Vasodilated guinea pig livers were perfused with normal erythrocytes and echinocytes suspended in isoviscous high- and low-molecular-weight dextran solutions. The relative flow resistance of these suspensions and the oxygen uptake of the livers were then determined. The relative flow resistance of the echinocytes that were suspended in high-molecular-weight dextran, however, was significantly higher than that of any other red cell suspension. The oxygen uptake was independent of the perfusion media. It is proposed that high-molecular-weight dextran induces echinocytes to attach to one another, and that this clumping together, and shape-transformation of red cells, hinders their flow in the vasodilated liver.     

Brightness versus apparent contrast. 3: Blurred disks and concentric cosine gratings.

Matching either the centre-brightness or the apparent contrast of incremental 1-deg disks, with varying edge-sharpness, yields quite different results. These suggest that the maximum brightness gradient at the edge determines apparent-contrast perception. However, no significant differences are found in matching the brightness maxima, the brightness minima, or the apparent contrast of concentric cosine gratings with varying spatial frequency. This means that spatial frequency influences both brightness extremes in the same way, and that apparent contrast is a function of the difference between the brightness extremes. The grating results therefore deviate from the results obtained with sharp as well as blurred disks. These contradictions complicate the construction of a model which unifies suprathreshold disk and grating perception.     

Light- and sucrose-dependent gene expression in photomixotrophic cell suspension cultures and protoplasts of rape (Brassica napus L.)

Light-dependent gene expression was analysed in photomixotrophic cell suspension cultures of rape (Brassica napus L.) growing in media containing either 2.0% or 0.6% sucrose. During growth in darkness phytochrome type I and NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) accumulated in both cell culture lines to a similar extent. Illumination with continuous white, blue or red light, but not with far-red light, resulted in disappearance of both chromoproteins within 24 h in both cell cultures. Further analysis showed that the phytochrome system of rape cell cultures reacts in a similar way to that of re-etiolated dicotyledonous plants, showing rapid P_fr destruction and rapid P_fr dark reversion. In contrast, the light-dependent expression of genes encoding the major chlorophyll a- and b-binding protein (CAB) and the re-accumulation of chlorophyll were found to be strongly dependent on sucrose concentration in culture media. Whereas cells grown in darkness in medium containing 2.0% sucrose showed, after exposure to continuous white light, a very weak re-induction of CAB mRNA, CAB protein and chlorophyll accumulation, the cells in medium containing 0.6% sucrose reacted very strongly. It was also possible to demonstrate that phytochrome (by high irradiance response, HIR, and by low fluence response, LF) and the blue/UV-A receptor are involved in the light-dependent gene expression of CAB. Similar to complete cells, protoplasts derived from the two different cell cultures showed an almost identical sucrose concentration-dependent and light-quality-dependent regulation of CAB mRNA accumulation. As the dark-grown photomixotrophic cells and protoplasts reflect some typical photoregulatory characteristics known from dark-grown plants it is supposed that this system will be an excellent tool for studying biochemical and molecular biological aspects of light-dependent signal transduction in cells of higher plants.     

Brightness versus apparent contrast. 1: Incremental and decremental disks with varying diameter.

This study describes the matching of the brightness and of the apparent contrast of foveal disks, presented as an increment or decrement with varying diameter against a 300 cd.m-2 background. If the brightness in the centre of the disks is matched with a constant reference brightness, the well-known spatial Broca-Sulzer phenomenon is obtained. This effect is not found if the apparent contrast of the disks is matched instead. All matching results and detection thresholds indicate that luminance increments and decrements are processed asymmetrically by the visual system: for decrements the threshold curve and curves of isobrightness as well as apparent iso-contrast are all shifted towards larger disk radii relative to those for increments. Two nonlinear single-channel models are tested against the data.     

The increase in viscosity and peroxidation of sarcoplasmic reticulum membrane lipids in isadrine myocarditis

The viscosity of membranes isolated from sarcoplasmic reticulum of rabbits with isadrine myocarditis was studied, using pyrene as a hydrophobic fluorescent probe. The increase in the viscosity of membranes from injured heart occurred at lower temperatures and was sharper than in the case of intact heart in both "free" and "bound" lipid domains. The increase in the lipid viscosity under myocarditis was associated with decreased Ca++, Mg++ -ATPase and cAMP-dependent protein kinase activities and with an elevated content of lipid peroxidation products.