Dark-adapted Frog Research Articles

Vitamin A and the role of the pigment epithelium during bleaching and regeneration of rhodopsin in the frog eye

The distribution of 11- cis and all- trans isomers of retinol and retinyl ester was investigated in the frog eye during light-and dark-adaptation (LA and DA). It was also established that freshly isolated rod outer segments (ROS) could regenerate their rhodopsin almost completely from added 11- cis retinol, provided that NADP was also present. The DA frog eye stores vitamin A equivalent to 120–230 mol% of the rhodopsin in the ROS. About 96% is in the RPE and is concentrated mainly in the oil-droplets (which can be separated by centrifugal flotation), the remaining 4% being split roughly equally between ROS and the rest of the retina. The oil-droplet vitamin A is virtually 100% esterified. Vitamin A occurs mainly as ester elsewhere, with the notable exception of the ROS, where the alcohol predominates. As much as 60% of this retinol can be 11- cis, and probably constitutes a pool of prosthetic groups for rhodopsin biosynthesis in the dark. In frogs dark-adapted (DA) for more than a day, more than half of the oil-droplet retinyl ester can be 11- cis. Since ROS lack an esterifying capability, all- trans retinol accumulates during LA of the isolated retina. If contact with the retinal pigment epithelium (RPE) is maintained, however, at least 87% of this retinol leaves the ROS, is esterified in the RPE and accumulates as all- trans retinyl ester in the oil-droplets. The 11- cis isomer is gradually consumed during LA and DA. The form in which vitamin A returns to the ROS has not been established, but some lines of evidence suggest that it is as the ester, mainly in the all- trans configuration. Isomerization to 11- cis probably takes place in the ROS, and during prolonged DA this appears to “leak” back to the RPE and cause a slow rise in the proportion of 11- cis over 20–30 hr in darkness (it should be noted that this effect cannot be accounted for by an influx of prosthetic groups from phagocytized ROS particles). Since the isolated retina has only a limited regenerative capacity, it is suggested that the ROS isomerase is specific for retinyl ester, which under normal circumstances can only be provided by the RPE.

Effect of light stimulation on the synthesis and release of GABA in rat and frog retinae

The effect of light stimulation on the incorporation of label from [ 14C]glucose and [ 3H]acetate into aspartate, glutamate, glutamine and GABA, both in vivo and in vitro, was studied in dark-adapted frog and rat retinae. With [ 14C]glucose as the precursor, but not with [ 3H]acetate, flashes of light appeared to increase the in vivo syntheses of both GABA and glutamine at the expense of glutamate in frog retina, while attenuating the labelling of aspartate and glutamate in isolated frog tissues. Amino acid synthesis from these radioactive substrates in rat retina was not altered under these conditions. Also, the turnover rates of amino acids derived from [ 14C]glucose in rat retina in vivo were closely similar in light and darkness. Isolated retinae from both species accumulated [ 14C]GABA from the incubation medium. In each case the subsequent release of this compound was augmented by electric field stimulation, high K + and by a high concentration of cold GABA in the medium, but not by a flashing light. Similarly, application of a light stimulus was unable to evoke the liberation of [ 14C]GABA derived from [ 14C]glucose in either species.

Spectral sensitivity of the frog eye in the ultraviolet and visible region

Spectral sensitivity of the dark-adapted frog's eye, eye cup and isolated retina was studied by means of an electroretinogram between 280 and 580 nm. Spectral sensitivity curve of isolated retina shows three peaks corresponding to α-, β- and protein absorption bands of the rhodopsin. Quantum yield of retinal excitation in the protein band, at 280 nm, is 0.45. Ocular media of Rana temporaria are transparent down to 330 nm so that the whole eye shows an appreciable sensitivity in the near ultraviolet. This fact may be of some ecological importance.

Autoradiographic and radiobiochemical studies on the incorporation of [6- 3H]glucosamine into frog rhodopsin

Our previous work on rhodopsin biosynthesis has dealt with the incorporation of amino acids into newly synthesized rhodopsin. The present work adds to this story by dealing with the intracellular site of incorporation of [ 3H]glucosamine into the polypeptide and the subsequent distribution of the glycoprotein within the cell. Freshly dissected retinas from dark-adapted frogs were incubated in the dark in oxygenated Ringer's bicarbonate medium containing d-[6- 3H]glucosamine. At selected intervals from 5 min to 6 hr, retinas were retrieved from the incubation medium and killed by aldehyde fixation. Following this, light and electron microscope autoradiograms of the tissue were prepared. In additional experiments, retinas were incubated for varying periods in a similar medium and rhodopsin was extracted, purified and analyzed for glucosamine incorporation. Glucosamine is first bound within the rod photoreceptor in the ribosome-rich myoid region, where the first glycosylation presumably occurs. By 20 min, the labeled product appears in the Golgi apparatus, where additional glycosylation presumably occurs. Finally, by 2 hr, the fully glycosylated product reaches the base of the outer segment, where it is inserted into the growing disc membrane. Biochemical analysis shows that the purified rhodopsin is heavily labeled by this time, and that the labeled precursor is incorporated chemically unchanged. The data imply that rhodopsin is sequentially glycosylated first on the ribosomes and then in the Golgi apparatus, and that the glycoprotein is complete in terms of its sugar moiety prior to its insertion into the growing disc membrane.

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

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.

Effects of background adaptation, length of exposure time and hypophysectomy on the quantity of melanophore-stimulating substances in the hypothalamo-hypophyseal complex of Rana pipiens

The quantity of the individual melanophore-stimulating substances (MSS's) was determined in aqueous extracts of the pars intermedia (PI), pars distalis (PD), and hypothalamus (H) of frogs which were light (L) or dark (D) adapted (A) for 2 or 8 days. The quantity of two of the substances common to the PI and PD was greater in LA than in DA frogs. In fact, one of the MSS's of the PI was not detectable in DA frogs. With the exception of the MSS which was not detectable, more MSS was present in the PI at day 8 than at day 2 for both DA and LA frogs. One MSS of the PD increased in quantily by day 8 when the frogs were light adapted, but did not increase in quantity after 8 days of dark adaptation. The second MSS of the PD was greater in quantity at day 8 than at day 2 in both DA and LA frogs. In both the PI and PD, the third MSS did not vary with background or length of exposure. The quantity of the single MSS in the H was greater in DA than LA frogs. There was more MSS in the H at day 8 than at day 2 in DA frogs, but in LA frogs the quantity did not change with length of exposure, nor was the MSS depleted. Hypophysectomy of frogs DA for 8 days reduced the quantity of MSS in the H to a level not significantly different from the level in LA frogs after 8 days of exposure. In conclusion, two of three MSS's of the PI and of the PD fluctuated in response to changes in background coloration and to the length of background exposure. The reduction in H content of MSS after hypophysectomy of dark-adapted frogs indicates that the major portion of H MSS originates in the hypophysis.

On the linear dichroism of frog rods

Single retinal rods from dark-adapted frog retinae were examined with the polarizing microscope in monochromatic illumination. Their retardance was determined as a function of the wavelength of the measuring light, of the bleaching light, and of the intensity of the bleaching light. The photosensitivity of bleaching was also determined. Illumination changed the retardance bimodally; it was shown to consist of two components of which the faster of the two is attributable to the linear dichroism of the rod resulting from the orientation of the rhodopsin molecules, and the other to the birefringence of the rod. During bleaching the linear dichroism due to rhodopsin is replaced for a few minutes before its disappearance by dichroism due to metarhodopsin III, which decays within 30 min after the onset of bleaching. The half-times of bleaching and of decay of dichroism can be made to agree if both components of the retardance are assumed to vary with illumination.

In vivo influence of light or darkness on the GABA system in the retina of the frog ( Rana pipiens)

An investigation of glutamate, γ-aminobutyric acid (GABA) and related enzyme systems in the frog retina has shown that adaptation to light and darkness has little or no effect on the content or localization of retinal glutamate. However, GABA levels are decreased by more than 40% in both the bipolar and ganglion cell layers of the dark-adapted frog retina. A decrease of glutamic carboxyl-lyase was also observed. GABA is concentrated in the frog retina primarily within cell layers which are rich in amacrine cells. The temporal decrease of retinal GABA and the decrease in threshold excitability during dark adaptation suggests a correlation between these two phenomena. Our observations are in agreement with the hypothesis that GABA in the frog retina is functionally related to inhibitory neural mechanisms.

Passive electrical properties of rod outer segments.

1. Measurements on a packed suspension of randomly oriented, dark-adapted frog rods at frequencies of 15 c/s-0.5 Mc/s indicate a behaviour similar to that of other biological materials.2. Results are analysed on the assumption that the low-frequency limiting resistance is determined by current flowing in the suspending medium and that, of the rods, two thirds are oriented perpendicular to the applied field and one third parallel to it. Those parallel to the field are treated as non-conductors.3. From the high-frequency limiting resistance the conductivity of the rod interior is calculated to vary linearly with the conductivity of the medium. The slope of the relation of internal to external conductivity is 0.50 with a limiting internal conductivity (at zero external) of 280 mumho/cm.4. On the assumption that the suspension can be represented as a single-capacitance network, the characteristic frequency of impedance is used to calculate a capacitance for the rod surface of 1.54 muF/cm(2). On the assumption of a distribution in properties of the suspension according to the theory of Bruggeman, the capacitance is calculated to have a value of about one half this.5. At frequencies below 5 kc/s the impedance locus deviates from the curve describing the behaviour at higher frequencies. It is suggested that this may involve conduction in a thin layer extending along the surface of the rod.

Fine structure of the frog electroretinogram

1. 1. Electroretinograms were elicited from dark-adapted frogs in response to stimuli of graded intensity and duration. 2. 2. The data support the hypothesis that the multiple peaks in the b-wave are caused by the summation of a number of sub-waves. 3. 3. Each sub-wave might be the result of some physiological process leading to the generation of an electrical potential.

Increase in sodium concentration of the isolated frog retina after light stimulation

The activity of Na 22 in the isolated, dark-adapted frog retina, after equilibration in frog Ringer's solution containing Na 22Cl, increased after light stimulation.

Kinetics of Rhodopsin Regeneration in the Eye of the Frog

RHODOPSIN, the red photosensitive pigment of rod vision, consists of the colourless protein, rod opsin, carrying 11-cisretinal (formerly retinene) as chromophore1. In the light, rhodopsin bleaches through orange-red intermediates (lumi- and meta-rhodopsin) into the yellow mixture of free all-trans retinal and opsin2. The enzyme alcohol dehydrogenase and DPNH2 in the rod outer segments reduce the retinal to retinol (formerly vitamin A)3. The all-trans retinol migrates from the rod outer segments into the pigment epithelium, where, most likely, an energy-utilizing process isomerizes the all-trans retinol into 11-cis retinol4. The exact material for the isomerization is, however, uncertain. This process supplies the pigment epithelium both in light- and dark-adapted frogs' eyes with a relatively great amount of 11-cis retinol. About 95 per cent of the retinol in the eye of the frog (Rana pipiens) is in the form of fatty acid-esters4.

The fine localization of nucleoside triphosphatase activity in the retina of the frog.

Nucleoside triphosphatase (NTPase) activity was demonstrated at the submicroscopic level in the frog retina by the Wachstein-Meisel method utilizing various purine and pyrimidine nucleosides. Under the electron microscope magnesium-activated NTPase was localized in the outer and inner segments, and in the plexiform layers. NTPase active sites in the cones were localized diffusely in the 70 to 80 A interspaces between the double membranes of the stacked lamellae and in the investing cytoplasm. In the rods, on the other hand, sites of activity were observed at the periphery of the stacked lamellae as discrete electron opaque deposits measuring 1000 to 1500 A which interdigitated between the lamellae for short distances. Deposits of reaction product appeared more numerous in rods of dark-adapted frogs stimulated with monochromatic light with a wave length of 510 mmicro. Enzyme activity was also observed in mitochondria of the rod and cone ellipsoids. In the outer and inner plexiform layers NTPase active sites were present on and between the membranes of axons and the plasma membranes of some of the neurons.

THE EXTRACTION AND ASSAY OF THE TYROSINASE FROM FROG SKIN: A STUDY ON THE INFLUENCE OF THE MELANOPHORE-STIMULATING HORMONE ON THE SYNTHESIS OF THE ENZYME

A soluble (microsomal) preparation of tyrosinase was obtained from frog skin (Rana pipiens) by pretreatment of strips of dorsal skin with trypsin. The relatively high activity of the enzyme towards tyrosine compared with that against dopa and other catechols would indicate that the enzyme is predominantly of the nature of a monophenolase. The rate of oxidation of D-tyrosine is about one-third that obtained with L-tyrosine. The activity of the enzyme (measured by oxygen consumption of the system) with dialyzed preparations could be increased up to fivefold by the addition of cupric ions. The enzyme is inhibited in the presence of copper-binding agents such as diethyldithiocarbamate. Highly purified specimens of intermedin had no effect on the production or turnover of tyrosinase in the frog skin, nor was there any effect on the tyrosinoxidase. There was little difference in the activity of the tyrosinase in light- or dark-adapted frogs, or in frogs which had received a series of injections of intermedin. The tyrosinase activity of the skin is subject to a large seasonal variation. The activity is about ten times greater during the period September to November than in the earlier period of the year.

THE EXTRACTION AND ASSAY OF THE TYROSINASE FROM FROG SKIN: A STUDY ON THE INFLUENCE OF THE MELANOPHORE-STIMULATING HORMONE ON THE SYNTHESIS OF THE ENZYME

A soluble (microsomal) preparation of tyrosinase was obtained from frog skin (Rana pipiens) by pretreatment of strips of dorsal skin with trypsin. The relatively high activity of the enzyme towards tyrosine compared with that against dopa and other catechols would indicate that the enzyme is predominantly of the nature of a monophenolase. The rate of oxidation of D-tyrosine is about one-third that obtained with L-tyrosine. The activity of the enzyme (measured by oxygen consumption of the system) with dialyzed preparations could be increased up to fivefold by the addition of cupric ions. The enzyme is inhibited in the presence of copper-binding agents such as diethyldithiocarbamate. Highly purified specimens of intermedin had no effect on the production or turnover of tyrosinase in the frog skin, nor was there any effect on the tyrosinoxidase. There was little difference in the activity of the tyrosinase in light- or dark-adapted frogs, or in frogs which had received a series of injections of intermedin. The tyrosinase activity of the skin is subject to a large seasonal variation. The activity is about ten times greater during the period September to November than in the earlier period of the year.

Acetylcholine in the Light and Dark Adapted Frog Retina

SummaryExtraction of frog retinas in acidified Ringer solution reveals an average Ach content of 3.4 γ/g in light-adapted and 3.6 γ/g in dark-adapted frogs. Direct comparison of Ach activity in extracts of lightand dark-adapted retinas, without reference to absolute magnitudes, also reveals no difference to absolute magnitudes, also reveals no difference greater than the error of the method. The lack of significant difference is in accord with the work of Therman, but not with that of Lange who finds a tenfold increase upon dark adaptation.

The electrical responses of dark‐adapted frogs' eyes to monochromatic stimuli

The Journal of PhysiologyVolume 88, Issue 4 p. 436-458 ArticleFree Access The electrical responses of dark-adapted frogs' eyes to monochromatic stimuli R. Granit, R. GranitSearch for more papers by this authorA. Munsterhjelm, A. MunsterhjelmSearch for more papers by this author R. Granit, R. GranitSearch for more papers by this authorA. Munsterhjelm, A. MunsterhjelmSearch for more papers by this author First published: 18 January 1937 https://doi.org/10.1113/jphysiol.1937.sp003452Citations: 54AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume88, Issue4January 18, 1937Pages 436-458 RelatedInformation

The electrical responses of light‐ and dark‐adapted frogs' eyes to rhythmic and continuous stimuli

The Journal of PhysiologyVolume 81, Issue 1 p. 1-28 ArticleFree Access The electrical responses of light- and dark-adapted frogs' eyes to rhythmic and continuous stimuli Ragnar Granit, Ragnar GranitSearch for more papers by this authorL. A. Riddell, L. A. RiddellSearch for more papers by this author Ragnar Granit, Ragnar GranitSearch for more papers by this authorL. A. Riddell, L. A. RiddellSearch for more papers by this author First published: 29 March 1934 https://doi.org/10.1113/jphysiol.1934.sp003112Citations: 118AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume81, Issue1March 29, 1934Pages 1-28 RelatedInformation