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V I Govardovskii

Publications and source records attributed to V I Govardovskii.

At least 19 recordsLinked to original sources
PubMed2006

Visual cycle and its metabolic support in gecko photoreceptors.

Photoreceptors of nocturnal geckos are transmuted cones that acquired rod morphological and physiological properties but retained cone-type phototransduction proteins. We have used microspectrophotometry and microfluorometry of solitary isolated green-sensitive photoreceptors of Tokay gecko to study the initial stages of the visual cycle within these cells. These stages are the photolysis of the visual pigment, the reduction of all-trans retinal to all-trans retinol, and the clearance of all-trans retinol from the outer segment (OS) into the interphotoreceptor space. We show that the rates of decay of metaproducts (all-trans retinal release) and retinal-to-retinol reduction are intermediate between those of typical rods and cones. Clearance of retinol from the OS proceeds at a rate that is typical of rods and is greatly accelerated by exposure to interphotoreceptor retinoid-binding protein, IRBP. The rate of retinal release from metaproducts is independent of the position within the OS, while its conversion to retinol is strongly spatially non-uniform, being the fastest at the OS base and slowest at the tip. This spatial gradient of retinol production is abolished by dialysis of saponin-permeabilized OSs with exogenous NADPH or substrates for its production by the hexose monophosphate pathway (NADP+glucose-6-phosphate or 6-phosphogluconate, glucose-6-phosphate alone). Following dialysis by these agents, retinol production is accelerated by several-fold compared to the fastest rates observed in intact cells in standard Ringer solution. We propose that the speed of retinol production is set by the availability of NADPH which in turn depends on ATP supply within the outer segment. We also suggest that principal source of this ATP is from mitochondria located within the ellipsoid region of the inner segment.

Animals↗
PubMed2006

Late stages of visual pigment photolysis in situ: cones vs. rods.

Slow photolysis reactions and the regeneration of the dark pigment constitute the mechanisms of dark adaptation whereby photoreceptor cells restore their sensitivity after bright illumination. We present data on the kinetics of the late stages of the photolysis of the visual pigment in intact rods and red- and green-sensitive cones of the goldfish retina. Measurements were made on single photoreceptors by means of a fast-scanning dichroic microspectrophotometer. We show that in cones the hydrolysis of the opsin-all-trans 3-dehydroretinal linkage proceeds with a half-time of approximately 5s at 20 degrees C that is almost two orders of magnitude faster than in rods. 3-Dehydroretinol in cones is produced approximately 3-fold faster than retinol in amphibian rhodopsin rods; the rate of the reaction is limited by the speed of retinal reduction catalyzed by retinoldehydrogenase. The fast hydrolysis of the 3-dehydroretinal/opsin Schiff base and the correspondingly fast appearance of the substrates for dark visual pigment regeneration (free opsin and 3-dehydroretinol) provide essential conditions for faster dark adaptation of cone (diurnal) as compared to rod (nocturnal) vision.

Animals↗
PubMed2005

Recombination reaction of rhodopsin in situ studied by photoconversion of "indicator yellow".

We measured the kinetics of recombination of 11-cis-retinal with opsin in intact frog rod outer segment (ROS). The rhodopsin in ROS was bleached and allowed to decay to "indicator yellow," a photoproduct where all-trans-retinal is partly free, and partly bound to non-specific amino groups of disk membranes. By briefly illuminating the "indicator yellow" by an intense 465 or 380-nm flash, we then photoconverted all-trans-retinal to (mostly) the 11-cis- form thus introducing into ROS a certain amount of cis-chromophore. The recombination of cis-retinal with opsin and the formation of rhodopsin were followed by fast single-cell microspectrophotometry. Regeneration proceeded with a time constant of approximately 3.5 min; up to 27% of bleached visual pigment was restored. The regenerated pigment consisted of 91% rhodopsin (11-cis-chromophore) and 9% of presumably isorhodopsin (9-cis-chromophore). The recombination of 11-cis-retinal with opsin inside the ROS proceeds substantially faster than rhodopsin regeneration in the intact eye and, hence, is not the rate-limiting step in the visual cycle.

Animals↗
PubMed2005

Two realms of dark adaptation.

The recovery of rod responsiveness after saturating flashes is greatly retarded above a certain critical level of rhodopsin bleaching (approximately 0.1%). A mathematical description of the process of turn-off of the phototransduction cascade allows attributing different phases of the recovery to specific products of rhodopsin photolysis. The fast phase is determined by quenching of metarhodopsin II and activated transducin. The slow phase is controlled by decay of partially inactivated (phosphorylated and arrestin-bound) metarhodopsins, and by regeneration of rhodopsin. The transition between the two regimes of adaptation is rather abrupt, occurring within a few-fold range of stimulus intensity. This marks the border between reversal of light adaptation and dark adaptation, as it is commonly defined.

Adaptation, Ocular↗
PubMed2001

Membrane protein diffusion sets the speed of rod phototransduction.

Retinal rods signal the activation of a single receptor molecule by a photon. To ensure efficient photon capture, rods maintain about 109 copies of rhodopsin densely packed into membranous disks. But a high packing density of rhodopsin may impede other steps in phototransduction that take place on the disk membrane, by restricting the lateral movement of, and hence the rate of encounters between, the molecules involved. Although it has been suggested that lateral diffusion of proteins on the membrane sets the rate of onset of the photoresponse, it was later argued that the subsequent processing of the complexes was the main determinant of this rate. The effects of protein density on response shut-off have not been reported. Here we show that a roughly 50% reduction in protein crowding achieved by the hemizygous knockout of rhodopsin in transgenic mice accelerates the rising phases and recoveries of flash responses by about 1.7-fold in vivo. Thus, in rods the rates of both response onset and recovery are set by the diffusional encounter frequency between proteins on the disk membrane.

Animals↗
PubMed2000

The gain of rod phototransduction: reconciliation of biochemical and electrophysiological measurements.

We have resolved a central and long-standing paradox in understanding the amplification of rod phototransduction by making direct measurements of the gains of the underlying enzymatic amplifiers. We find that under optimized conditions a single photoisomerized rhodopsin activates transducin molecules and phosphodiesterase (PDE) catalytic subunits at rates of 120-150/s, much lower than indirect estimates from light-scattering experiments. Further, we measure the Michaelis constant, Km, of the rod PDE activated by transducin to be 10 microM, at least 10-fold lower than published estimates. Thus, the gain of cGMP hydrolysis (determined by kcat/Km) is at least 10-fold higher than reported in the literature. Accordingly, our results now provide a quantitative account of the overall gain of the rod cascade in terms of directly measured factors.

Animals↗
PubMed1999

Inhibition of membrane-bound carbonic anhydrase decreases subretinal pH and volume.

PURPOSE: The lipophilic carbonic anhydrase (CA) inhibitor acetazolamide has been shown to enhance subretinal fluid resorption, reduce subretinal pH, and can improve cystoid macular edema, but its clinical use is limited by systemic side effects. While these are most likely a result of inhibiting intracellular CA isoenzymes, retinal pigment epithelial (RPE) transport is thought to be modulated via membrane-bound CA. This study investigates whether benzolamide, a hydrophilic CA inhibitor that does not readily penetrate cell membranes, is sufficient to modulate subretinal volume and pH. METHODS: Volume and pH were assessed in the subretinal space (SRS) of the perfused chick retina-RPE-choroid preparation by calculating these variables from data obtained with two different double-barreled, ion-selective electrodes (H+ for pH and the extracellular space marker tetramethylammonium (TMA+) for SRS volume). Light induced variations and changes in baseline measurements were recorded before and after addition of 10(-4) M acetazolamide or benzolamide to the basal perfusion. RESULTS: Basal perfusion with either drug induced both an acidification of the SRS by 0.02-0.04 pH units, which occurred within 60 s, as well as an increase in the amplitude of the light-induced alkalinisation of the SRS. TMA+ concentration in the SRS increased steadily over a period of several minutes after basal perfusion with either of the CA inhibitors, and the calculated SRS volume was reduced by 40% within 8-10 min. CONCLUSION: The observation that benzolamide had effects equal to acetazolamide suggests that inhibition of membrane-bound CA at the basolateral membrane of the RPE is sufficient to decrease subretinal pH and volume. This may represent a clinically important mechanism for the resorption of sub- and intraretinal fluid.

Acetazolamide↗
PubMed1998

Photoreceptor coupling and boundary detection.

Electrical coupling between photoreceptors results in the extensive spreading of output potentials along the syncytium of photoreceptor terminals. This smoothing of output potentials seems to make spatial resolution worse. However, the photoreceptor noise that is considered to be non-correlated both in space and time is smoothed to the greater extent than the correlated potential difference across the boundaries between areas of different brightness. This improves the signal-to-noise ratio more for more extended boundaries and favours lowering the threshold so that they can be detected more easily during the subsequent processing. The results have a striking parallel with a well known dependence of contrast threshold on stimulus size as measured psychophysically.

Contrast Sensitivity↗
PubMed1997

The photoreceptors and visual pigments of the garter snake (Thamnophis sirtalis): a microspectrophotometric, scanning electron microscopic and immunocytochemical study.

Scanning electron microscopy, immunocytochemistry, and single cell microspectrophotometry were employed to characterize the photoreceptors and visual pigments in the retina of the garter snake, Thamnophis sirtalis. The photoreceptor population was found to be comprised entirely of cones, of which four distinct types were identified. About 45.5% of the photoreceptors are double cones consisting of a large principal member joined near the outer segment with a much smaller accessory member. About 40% of the photoreceptors are large single cones, and about 14.5% are small single cones forming two subtypes. The outer segments of the large single cones and both the principal and accessory members of the doubles contain the same visual pigment, one with peak absorbance near 554 nm. The small single cones contain either a visual pigment with peak absorbance near 482 nm or one with peak absorbance near 360 nm. Two classes of small single cones could be distinguished also by immunocytochemistry and scanning electron microscopy. The small single cones with the 360-nm pigment provide the garter snake with selective sensitivity to light in the near ultraviolet region of the spectrum. This ultraviolet sensitivity might be important in localization of pheromone trails.

Animals↗
PubMed1994

Visual pigments and the photic environment: the cottoid fish of Lake Baikal.

The endemic cottoid fish of Lake Baikal in Eastern Siberia offer a singular opportunity for examining within a number of closely related species, the relationships of visual pigments, photoreceptor complements and depth within a deep freshwater environment. The lake, the deepest (1600 m) and one of the largest and most ancient in the world, is unique in that the oxygen levels at the bottom are only reduced to about 80% of the surface levels. We have studied, by light microscopy, microspectrophotometry and visual pigment extraction, the retinas from 17 species of Baikal cottoids that live at different depths within the lake. Generally the retinas contain, in addition to rods, large green-sensitive double cones and small blue-sensitive single cones: surprisingly for freshwater fish, the visual pigments are based on Vitamin A1. The lambda max of both rods and cones are displaced to shorter wavelengths with increasing depth. Surface species have cones with lambda max at about 546, 525 and 450 nm and rods at 523 nm, deeper living species retain cones, but with lambda max shifting towards 500 and 425 nm and with rods at 480 nm, whereas the deepest living fish possess only rods (lambda max 480-500 nm). These data clearly show a correlation between photoreceptor complement, visual pigment lambda max and depth, but question the hypothesis that there is a correlation of pigment lambda max with water colour since, in contrast to oceanic waters, the maximum transmission of Baikal water is between 550 and 600 nm.

Animals↗
PubMed1994

Response univariance in bull-frog rods with two visual pigments.

Rods in the bull-frog retina contain varying proportions of rhodopsin (lambda max = 502 nm) and porphyropsin (lambda max = 527 nm) in a dorso-ventral gradient from the porphyropsin-rich dorsal rim to the virtually pure rhodopsin fields of the central and ventral retina. We investigated if quantal excitations in the same rod are different depending on whether they are initiated by isomerization of a rhodopsin or a porphyropsin molecule. Current photoresponses were recorded from dark-adapted rods by sucking the outer segment into a recording pipette. The relation between pigment composition and spectral sensitivity was established by comparison with microspectrophotometrically measured absorbance spectra of rods from the same neighbourhood. Rods with suitable porphyropsin: rhodopsin mixtures (ideally between 1:4 and 1:2) were stimulated with flashes of red (608 nm) and blue (465 nm) light, whereby the red light will isomerize porphyropsin much more often than rhodopsin, and the reverse will be true of the blue light. The amplitude and shape of the single-photon response were found to be identical for the "red" and "blue" flash series to within measurement error (ca 10%). This indicates that the quantal responses initiated by the two pigments are identical.

Animals↗
PubMed1994

Mathematical model of TMA+ diffusion and prediction of light-dependent subretinal hydration in chick retina.

PURPOSE: To derive a mathematical model of TMA+ diffusion across the retina that can be used to estimate the amplitude and kinetics of the light-evoked increase in subretinal hydration and its effect on the concentration of other ions. METHODS: All experimental data were obtained in chick retina-pigment epithelium-choroid preparations as described in the accompanying paper. RESULTS: Diffusional properties of the retina were derived from the time course of [TMA+]o in the subretinal space (SRS) after changes in the retinal perfusate. Then, the SRS volume changes underlying the light-induced [TMA+]o response can be derived using a mathematical model of TMA+ diffusion. Complete retinal depth series of light-evoked [TMA+]o responses could be simulated by producing a corresponding expansion of the SRS. Volume changes inferred from the diffusion model were 2.2 to 3.8 times larger and more prolonged than could be derived directly from delta [TMA+]o. The model predicted up to a 20% peak increase in subretinal-space hydration during illumination. The effects of this volume increase on subretinal K+ and Ca2+ were estimated. These predictions were supported by inhibiting the volume increase with DIDS, which blocks retinal pigment epithelium basal membrane Cl- conductance. CONCLUSIONS: The primary source of light-evoked changes in extracellular TMA+ concentration recorded throughout the retina is an increase in hydration (volume) of the subretinal space. The response spreads to the inner retina by diffusion. Effects of TMA+ diffusion lead to large underestimates of the underlying volume changes. The light-evoked volume change alters the composition of the subretinal space and light-induced responses of other ions.

Animals↗
PubMed1992

Cones in the retina of the Mongolian gerbil, Meriones unguiculatus: an immunocytochemical and electrophysiological study.

Immunocytochemistry revealed in the retina of the Mongolian gerbil three immunologically distinct photoreceptor cell types. Rods comprising about 87% of the total receptor population were selectively recognized by an antirhodopsin serum (AO). The most abundant cone type (11-13% of photoreceptors) was labeled by the monoclonal antibody COS-1, specific in mammals to the middle-to-long-wave sensitive cone visual pigments. A minor cone population (2.5-5% of the cones) reacted with the monoclonal antibody OS-2, shown earlier to bind to the blue cones in mammalian species. Color substitution experiments revealed on the ERG level a color discrimination capability which must be attributed to the cooperative activity of green-sensitive (COS-1 positive) and blue-sensitive (OS-2 positive) cones. We conclude that the Mongolian gerbil has a well developed cone system, and that it may possess dichromatic green-blue color vision.

Animals↗
PubMed1992

Immunocytochemical reactivity of rod and cone visual pigments in the sturgeon retina.

Microspectrophotometry and immunocytochemistry with several antivisual pigment antibodies were used to study visual cells of the Siberian sturgeon, Acipenser baeri Brandt. The retina contained rods and three morphological types of cones: large cones with oil drops, small cones with oil drops, and cone-like cells without oil drops. Rods and cone-like drop-free cells were found to possess porphyropsin-549, while the large oil drop-bearing cones contained red-sensitive (P613), green-sensitive (P542), and blue-sensitive (P462) visual pigments. The immunocytochemical staining pattern with three antibodies to visual pigment proteins also revealed one visual pigment in rods and three visual pigments in cones. Rods were labeled with all three antibodies, while the majority of large cones (type I), presumably the red-sensitive ones, were negative with the polyclonal serum AO against bovine opsin. A less-frequently occurring large cone type (type II) was stained by all three antibodies including mAb COS-1 specific to middle-to-long-wave visual pigments in birds and mammals, and is thought to be green-sensitive. An even less-frequent large cone type (type III, probably the blue-sensitive one) did not bind COS-1. The small cones with oil droplets showed immunoreactivities similar to either type II or type III cones. The oil drop-free small photoreceptor exhibited a staining pattern identical with that of rods. These results indicate that the immunocytochemical approach can be used to reveal photoreceptor-specific neural connections in the sturgeon retina.

Animals↗
PubMed1991

Spectral characteristics of photoreceptors and horizontal cells in the retina of the Siberian sturgeon Acipenser baeri Brandt.

In the retina of Siberian sturgeon, three spectral classes of photoreceptors were identified by microspectrophotometry. These were rods, oil drop-containing and oil drop-free cones possessing P549, and oil drop-containing cones possessing P613 and P465. With intracellular recordings, rod-driven, cone-driven, and mixed horizontal cells of luminosity type were found, as well as color-opponent horizontal cells of, at least, 6 kinds. Biphasic R/G cells received hyperpolarizing input either from rods or from green cones; in R/B cells, it was from blue cones. Other three types comprised biphasic G/B (or Y/B), RG/B, and triphasic G/BR cells. So the Chondrostean retina has the color-processing circuitry common for all ray-finned fishes.

Action Potentials↗
PubMed1990

The frequency of isomerization-like 'dark' events in rhodopsin and porphyropsin rods of the bull-frog retina.

1. The dark current and responses to dim flashes were recorded with the suction pipette technique from single rods in pieces of bull-frog retina taken from either the dorsal porphyropsin or the ventral rhodopsin field. 2. The composition of visual pigment in the rods was determined by microspectrophotometry. Rods from the dorsal pieces contained 70-88% porphyropsin523 mixed with rhodopsin502. The ventral rods contained almost pure rhodopsin, any possible admixture of porphyropsin being below the level of detectability (less than 5%). 3. In most cells, the responses to dim flashes were well fitted by a four-stage linear filter model, with no systematic differences in the response kinetics of porphyropsin and rhodopsin rods. The amplitude of saturated responses varied between 8 and 55 pA and that of responses to single isomerizations between 0.4 and 3.5 pA. 4. In porphyropsin rods, discrete events similar to the response to one photoisomerization were clearly seen in complete darkness. The dark current amplitude histogram was fitted by a convolution of the probability densities for the Gaussian continuous noise component and the averaged dim-flash response waveform. This allows estimation of the frequency and amplitude of discrete events and the standard deviation of the continuous component. The mean frequency of discrete dark events thus obtained from six porphyropsin cells was 0.057 rod-1 s-1 at 18 degrees C. 5. In rhodopsin rods, the dark current amplitude histogram appeared completely symmetrical, indicating that the frequency of discrete events must be lower than 0.005 rod-1 s-1 (except in one rod where it was 0.006 events rod-1 s-1). Per molecule of rhodopsin, the events are then at least 5 times rarer than reported for toad rhodopsin rods at the same temperature. 6. The low rate of isomerization-like 'dark' events in bull-frog rhodopsin rods shows, firstly, that results cannot be generalized across species even for rhodopsins which appear spectrally identical. Secondly, it suggests that these events need not (in an evolutionary sense) constitute an irreducible noise factor which must set the ultimate limit to the sensitivity of dark-adapted vision. 7. The difference between porphyropsin and rhodopsin rods shows that, given (presumably) the same opsin, the pigment utilizing retinal2 and absorbing maximally at longer wavelengths produces more noise. The signal/noise ratio attained in the photoreceptor may be an important factor in the natural selection of visual pigments.

Animals↗
PubMed1975

[Some quantitative characteristics of the frog retinal rod outer segments].

Retinal rod outer segments in frogs were studied by means of light microscopy, refractometry, microspectrophotometry, and electron microscopy. Analysis of the data obtained shows that an unidentified substance, which makes up about 50% of outer segment dry weight, is lost during routine biochemical investigations. The protein parts of the rhodopsin molecules make up 85% of the outer segments proteins and 25% of outer segment dry weight. Rhodopsin molecules can be arranged in a square array with a unit cell side of about 7 nm on one side of each disk membrane. Lipids in a single membrane occupy only 2 nm, and disk membranes are strongly hydrated.

Animals↗