Biomedical subjects

D R Pepperberg

Publications and source records attributed to D R Pepperberg.

At least 37 records · Page 2Linked to original sources

PubMed1993

Interphotoreceptor retinoid-binding protein (IRBP). Molecular biology and physiological role in the visual cycle of rhodopsin.

The regeneration of visual pigment in rod photoreceptors of the vertebrate retina requires an exchange of retinoids between the neural retina and the retina pigment epithelium (RPE). It has been hypothesized that interphotoreceptor retinoid-binding protein (IRBP) functions as a two-way carrier of retinoid through the aqueous compartment (interphotoreceptor matrix) that separates the RPE and the photoreceptors. The first part of this review summarizes the cellular and molecular biology of IRBP. Work on the IRBP gene indicates that the protein contains a four-fold repeat structure that may be involved in binding multiple retinoid and fatty acid ligands. These repeats and other aspects of the gene structure indicate that the gene has had an active and complex evolutionary history. IRBP mRNA is detected only in retinal photoreceptors and in the pineal gland; expression is thus restricted to the two photosensitive tissues of vertebrate organisms. In the second part of this review, we consider the results obtained in experiments that have examined the activity of IRBP in the process of visual pigment regeneration. We also consider the results obtained on the bleaching and regeneration of rhodopsin in the acutely detached retina, as well as in experiments testing the ability of IRBP to protect its retinoid ligand from isomerization and oxidation. Taken together, the findings provide evidence that, in vivo, IRBP facilitates both the delivery of all-trans retinol to the RPE and the transfer of 11-cis retinal from the RPE to bleached rod photoreceptors, and thereby directly supports the regeneration of rhodopsin in the visual cycle.

Animals↗

PubMed1992

Hydroxylamine-dependent inhibition of rhodopsin phosphorylation in the isolated retina.

Hydroxylamine (NH2OH), a substance known to accelerate the decay of the metarhodopsin II bleaching intermediate of rhodopsin, was examined for its effect on the light-dependent phosphorylation of rhodopsin in the intact, isolated retina. Groups of ovine and bovine retinas that had been pre-incubated in darkness with 32P-inorganic phosphate were supplemented with NH2OH at final concentrations of up to 20 mM, then irradiated and further incubated in darkness. Rod outer segments isolated from the incubated retinas were subjected to SDS-PAGE; the gel was analysed for 32P (autoradiography) and protein (Coomassie staining), to determine the specific radioactivity (ratio of 32P and protein levels; '32P/opsin') of the opsin monomer band. Among retinas of a given experimental group, 32P/opsin declined with increasing concentration of added NH2OH. The relative value of 32P/opsin exhibited by controls (0 mM NH2OH) was halved in the presence of about 1-2 mM NH2OH, and was reduced by greater than or equal to 80% in the presence of 20 mM NH2OH. Supplementation of the retina with 20 mM NH2OH 1 min after irradiation caused relatively little reduction in 32P/opsin. The results indicate that the light-dependent phosphorylation of rhodopsin in situ is substantially inhibited by NH2OH at millimolar levels. The data are discussed in relation to previous electrophysiological studies that have examined rod dark adaptation in NH2OH-treated retinas.

Animals↗

PubMed1992

Light-dependent delay in the falling phase of the retinal rod photoresponse.

Using suction electrodes, photocurrent responses to 100-ms saturating flashes were recorded from isolated retinal rods of the larval-stage tiger salamander (Ambystoma tigrinum). The delay period (Tc) that preceded recovery of the dark current by a criterion amount (3 pA) was analyzed in relation to the flash intensity (If), and to the corresponding fractional bleach (R*0/Rtot) of the visual pigment; R*0/Rtot was compared with R*s/Rtot, the fractional bleach at which the peak level of activated transducin approaches saturation. Over an approximately 8 ln unit range of I(f) that included the predicted value of R*s/Rtot, Tc increased linearly with ln I(f). Within the linear range, the slope of the function yielded an apparent exponential time constant (tau c) of 1.7 +/- 0.2 s (mean +/- S.D.). Background light reduced the value of Tc measured at a given flash intensity but preserved a range over which Tc increased linearly with ln I(f); the linear-range slope was similar to that measured in the absence of background light. The intensity dependence of Tc resembles that of a delay (Td) seen in light-scattering experiments on bovine retinas, which describes the period of essentially complete activation of transducin following a bright flash; the slope of the function relating Td and ln flash intensity is thought to reflect the lifetime of photoactivated visual pigment (R*) (Pepperberg et al., 1988; Kahlert et al., 1990). The present data suggest that the electrophysiological delay has a similar basis in the deactivation kinetics of R*, and that tau c represents TR*, the lifetime of R* in the phototransduction process. The results furthermore suggest a preservation of the "dark-adapted" value of TR* within the investigated range of background intensity.

Ambystoma↗

PubMed1991

Depalmitylation with hydroxylamine alters the functional properties of rhodopsin.

Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).

Animals↗

PubMed1991

Functional properties of interphotoreceptor retinoid-binding protein.

It has been hypothesized that interphotoreceptor retinoid-binding protein (IRBP) functions as a two-way carrier of retinoid between the retinal pigment epithelium (RPE) and rod photoreceptors in the vertebrate eye. This hypothesis has been tested in recent studies that have employed purified, initially ligand-free, bovine IRBP and the "RPE-eyecup" obtained from the toad (Bufo marinus) eye. The present experiments further characterize the IRBP/RPE-eyecup system with respect to (i) the solubilization and protection of retinol by IRBP, and (ii) the time course of IRBP-mediated release of 11-cis retinal by the RPE. The data, together with previous findings in the IRBP/RPE-eyecup preparation, support the view that 11-cis retinal is the principal retinoid released by the RPE into IRBP-supplemented aqueous medium, and that IRBP in vivo promotes the regeneration of rhodopsin by facilitating the exchange of retinoid between bleached rods and the RPE.

Animals↗

PubMed1990

Effect of bleached rhodopsin on signal amplification in rod visual receptors.

Bleaching of rhodopsin markedly desensitizes the vertebrate visual system during a subsequent period of dark adaptation. Previous studies have indicated an origin of bleaching desensitization in the visual pigment itself, but have not identified the mechanism of action. A candidate for the site at which densensitization is initially expressed is the activation of transducin (formation of T*) on the rod disk membranes; this reaction directly involves rhodopsin in its photoactivated (R*) form and mediates initial amplification of the visual signal (reviewed in refs 7-9). We have analysed the effect of bleaching on the sensitivity of a flash-induced light-scattering signal known to monitor the disk-based amplifier, and which has been established as specifically monitoring transducin activation. We have recorded this signal from functioning retinal rods in situ ('ATR' signal) and find that bleaches inducing a pronounced, sustained loss in rod electrophysiological sensitivity do not alter the sensitivity of the ATR response after correction for reduced quantum catch. Our results indicate that the biochemical gain of the R*----T* transduction stage remains unchanged in the presence of bleached pigment and implicate a subsequent reaction as the first to show a sustained, bleaching-dependent gain reduction.

Animals↗

PubMed1990

Interphotoreceptor retinoid-binding protein promotes rhodopsin regeneration in toad photoreceptors.

Interphotoreceptor retinoid-binding protein (IRBP) has been hypothesized to function as an intercellular shuttle in the vertebrate eye, serving to transport retinoids between the retinal pigment epithelium (RPE) and photoreceptors in the process by which visual pigment is regenerated after photolysis. This hypothesis was tested in preparations utilizing the toad (Bufo marinus) eye and purified, initially ligand-free IRBP obtained from the bovine eye. Rod outer segments (ROS) or neural retinas were isolated and bleached, then incubated with native RPE (RPE-eyecup) in the presence or absence of IRBP. The amount of rhodopsin present after incubation was determined by spectrophotometric analysis and compared with that in control preparations receiving bovine serum albumin or Ringer's solution only. Supplementation with IRBP enhanced the formation of rhodopsin in both the ROS/RPE-eyecup and retina/RPE-eyecup preparations. Regeneration in ROS/RPE-eyecups receiving IRBP (1.8 nmol) increased in a roughly linear manner with the period of incubation (0-4 hr), at a rate of 0.44 nmol/hr. The extent of regeneration was graded with the quantities of IRBP and opsin introduced into the RPE-eyecup. With increasing amounts of IRBP (up to 5.2 nmol) or of initially available opsin (up to 15.6 nmol), the amount of rhodopsin formed (3-hr incubation) approached the same plateau value, about 2.5 nmol. Analysis of IRBP-supplemented Ringer's solution incubated in the RPE-eyecup showed 11-cis-retinal to be virtually the only retinoid withdrawn from the RPE. With large quantities of IRBP (3.2-9.2 nmol), the amount of 11-cis-retinal (2.7 +/- 0.5 nmol) withdrawn from the RPE during a 3-hr incubation was similar to the plateau value of rhodopsin formed in the ROS/RPE-eyecup. No 11-cis-retinal was observed in albumin-supplemented Ringer's solution (0.4-11.2 nmol of bovine serum albumin) or in Ringer's alone after similar incubation in the RPE-eyecup. The results suggest that an IRBP-mediated transfer of 11-cis-retinal from the RPE to the rods supports rhodopsin regeneration in vivo.

Animals↗

PubMed1990

Dark adaptation in locally detached retina.

Nonrhegmatogenous retinal detachments were formed in the eyes of Dutch rabbits by subretinal injection of Hanks' balanced salt solution. The electroretinogram (ERG) was recorded locally from the acutely detached retina, and simultaneously from the surrounding attached retina (vitreal ERG [VERG]), before and after exposure to diffuse intense irradiation. Light adaptation elevated b-wave threshold for both the local ERG (LERG) and VERG by about 3 log units; thresholds for both responses recovered fully within 60-90 min after the irradiation. The normal time course of dark adaptation of the LERG suggests the occurrence of substantial rhodopsin regeneration in the rod photoreceptors of nonrhegmatogenously detached retina. These results differ from reports that visual pigment regeneration is slow in central serous chorioretinopathy, possibly because our detachments were studied within hours of formation, whereas some photoreceptor degeneration may be present in older clinical detachments.

Animals↗

PubMed1989

Interphotoreceptor retinoid-binding protein: role in delivery of retinol to the pigment epithelium.

The ability of interphotoreceptor retinoid-binding protein (IRBP) to facilitate the incorporation of retinol into retinyl esters by the retinal pigment epithelium (RPE) was examined in toad (Bufo marinus) eyecup preparations devoid of neural retina (RPE-eyecup). Solutions containing purified bovine IRBP and all-trans[3H]retinol were introduced into the vitreal cavity of the RPE-eyecup. After incubation at 22 degrees C, [3H]retinyl ester was extracted from the RPE cells and isolated by high performance liquid chromatography. All-trans[3H]retinyl ester formed in the RPE increased with time of incubation (up to 2 hr) and with concentration of IRBP (up to 10 microM). The increase with IRBP concentration accompanied, and presumably resulted from, an increased transfer of [3H]retinol to the RPE-eyecup. With higher concentration of IRBP (20-30 microM), both the amount of [3H]retinyl ester formed (relative to the peak value at 10 microM IRBP) and the overall molar content of endogenous retinyl ester were reduced. On the other hand, bovine serum albumin at relatively high concentration (90 microM) was less effective than 3 microM IRBP in supporting the formation of [3H]retinyl ester, and it did not reduce the level of native retinyl ester in the RPE. Using 3 microM IRBP, levels of [3H]retinyl ester formed were comparable to or exceeded those obtained with phosphatidyl choline (0.9 mg ml-1) or serum retinol-binding protein (3 microM). The data are consistent with the hypothesized role of IRBP as a carrier of retinol between the retina and RPE in the operation of the visual cycle.

Animals↗

PubMed1989

Activity of rhodopsin in vitamin A-deprived rats: light-dependent binding of G-protein.

Levels of rhodopsin in the photoreceptors of Long-Evans rats were reduced by approximately 55% through dietary deprivation of vitamin A. The interaction of visual pigment with G-protein was examined in receptor outer segment (ROS) membranes obtained from these animals. A binding assay was used to quantitate affinity of the visual pigment in unbleached and bleached ROS membranes for the alpha and beta subunits of exogenous G-protein. Extents of binding were similar to those observed for ROS membranes of rats raised on a normal (vitamin A-supplemented) diet. The results are consistent with a normal capacity for G-activation by photoactivated rhodopsin (R*) in vitamin A-deprived animals. They further indicate that "free opsin" arising in vitamin A deficiency, unlike R*, has relatively low affinity for G-protein.

Animals↗

PubMed1988

Photic modulation of a highly sensitive, near-infrared light-scattering signal recorded from intact retinal photoreceptors.

On stimulation by green flashes, the isolated, aspartate-treated bovine retina exhibits transient changes in the scattering of near-infrared (880 nm) light. A single component, termed the "ATR" (a flash-induced scattering signal, where ATR designates amplified transient-retina), dominates the amplitude and rising-phase kinetics of the initial peak of the light-scattering response. Superfusion with physiological solution containing low Na+ concentration reversibly abolishes the photoreceptor electroretinographic response but preserves the ATR signal, indicating a receptoral origin for the ATR. The increase of ATR amplitude (A/Amax) with flash intensity (R*/R, where R indicates rhodopsin) is described by A/Amax = (1- e-kR*/R), with R*/R = k-1 occurring on generation of approximately two photoactivated rhodopsins (R*s) per disc surface in the rod outer segment. Weak background light and bright flashes reversibly depress the ATR. Kinetic and sensitivity data suggest a basis of the ATR in stochastic, unit activation events, each initiated by a single R*. They further suggest an essential invariance of the unit event under differing conditions of illumination. A delay, apparently governed by the lifetime of a light-activated substance regulating ATR generation, precedes ATR recovery after a bright flash. The flash dependence of the delay period indicates an upper limit of 3 s for the lifetime of R* in the ATR-generating process. The unit event appears to be an R*-catalyzed and disc-localized reaction of phototransduction.

Animals↗

PubMed1988

Immunolocalization of 48K in rod photoreceptors. Light and ATP increase OS labeling.

An abundant, light-modified protein of Mr approximately equal to 51 kD, homologous to a previously described protein (termed 48K, arrestin, or S-antigen) of bovine retina, was identified and characterized in rod photoreceptors of the toad (Bufo marinus). Isolated, intact retinas were incubated in darkness, or irradiated under physiological conditions [dark-adapted (DA) and light-adapted (LA) retinas]. Using polyclonal antibodies raised in rabbit against purified toad 48K, and post-embedding immunoelectron microscopy (second antibody conjugated with 5 nm gold particles), we examined the localization of 48K in DA vs. LA rods. Physiological incubations and early fixation steps were carried out in the presence vs. absence of ATP (40 microM), a substance thought to support the activity of 48K in vivo. The distribution of 48K in rods was analyzed by quantitating the density of gold label within subcellular compartments. In DA rods, labeling was highest in the myoid region of the inner segment. Light adaptation increased 48K immunoreactivity within the outer segment, and decreased labeling within calycal processes. Labeling in outer segments of LA rods was maximal in the basal region. Treatment with ATP both amplified the light-dependent increase in basal OS labeling, and augmented the nonuniformity of basal vs. apical labeling in LA ROS. Morphometric analysis of labeling density over the length of DA vs. LA ROS indicated an approximately equal to 1.5-fold net increase in 48K label in LA ROS. By comparison, biochemical analysis of 48K level indicated an approximately equal to 1.8-fold increase in 48K in LA preparations. Together, the biochemical and immunocytochemical data indicate that the relative amount of 48K is increased in LA ROS, not just its immunoreactivity. The data are consistent with a selective movement of 48K, a cytoplasmic ROS protein, into ROS during LA.

Adaptation, Physiological↗

PubMed1987

Photoreceptor processes in visual adaptation.

In this paper we have stressed two experimental results in need of explanation: (i) the reduced efficacy with which (remaining, abundant) rhodopsin in the light-adapted receptor mediates the flash response; and (ii) the disparity in conditions of irradiation (weak background vs. extensive bleaching) leading to equivalent conditions of threshold. The model discussed above suggests, in molecular terms, a possible basis for both properties of receptor adaptation. On the view developed here, property (i) derives from the ability of photoactivated or bleached pigment (R or B) to restrict dramatically the availability of a substance required for phototransduction. Property (ii) derives in large part from the pronounced disparity in the effectiveness of R (during illumination) and B (remaining after illumination) in reducing the availability of this substance. On this view, the "equivalence" of threshold elevation in states of light- vs. dark-adaptation derives from an overall equality of a product of factors (Q, Etot/Es, and J of equation 2). Under all but extreme conditions, this aggregate of factors is dominated by the term Etot/Es, reflecting the functional state of E.

Animals↗

PubMed1987

Interaction of visual pigment with G-protein: effects of bleaching in native and reconstituted ROS preparations.

Native and reconstituted preparations of bullfrog rod outer segment (ROS) membranes were analysed for the binding of the alpha and beta subunits of G-protein. Following incubation at 22-28 degrees C under varying conditions of illumination and chemical treatment, preparations were chilled and extracted with hypotonic medium lacking-vs.-containing GTP (H and HG extracts, respectively). The extent of binding of G alpha,beta ('percentage G bound') was determined by measurement of the relative abundance of G alpha, beta in the H and HG extracts. The addition of G to unbleached, G-depleted membranes (D-ROS) yielded relatively little binding of the G (14 +/- 13%). G reintroduced at congruent to 1 min vs. congruent to 90 min after major bleaching yielded, respectively, levels of binding of 88 +/- 8% and 24 +/- 8%. Native G, extracted at congruent to 90 min post-bleach and added to freshly bleached D-ROS, exhibited binding exceeding that in the parent (bleached +90 min) sample. Supplementation of reconstituted suspensions with all-trans retinal, and subsequent incubation in darkness, yielded 30-35% G-binding; similar treatment with 11-cis retinal yielded binding in the range of 0-32%. Upon irradiation, suspensions previously supplemented with either isomer exhibited approximately 60% binding. Incubation with 11-cis retinal had no substantial effect on the condition of tight binding observed shortly after bleaching. The data are discussed in relation to previous studies of visual pigment bleaching and regeneration.

Animals↗

PubMed1987

Localization of retinal "48K" (S-antigen) by electron microscopy.

The subcellular localization of the 48-kDa protein, a soluble protein in rod photoreceptors that is identical to retinal S-antigen, has been examined in photoreceptors of the toad (Bufo marinus) by immunoelectron microscopy. Pairs of retinas, exposed to controlled conditions of light- and dark-adaptation were compared. Lowicryl-embedded sections of light-adapted versus dark-adapted retinas were incubated with polyclonal anti-48K antibodies, then post-treated with second antibodies that were conjugated with 5 nm gold particles. In dark-adapted rod photoreceptors, the highest labeling density (approximately 39 gold particles/micron2) was found in the myoid region of the inner segment. Light-adaptation significantly affected the pattern of labeling. The principal change was a two-fold increase in labeling density in the basal region of the outer segment; this was accompanied by a decrease in labeling density in the myoid region. Labeling densities in the other subcellular compartments examined ranged from 7-10 gold particles/micron2; these densities did not differ substantially between light- versus dark-adapted preparations.

Adaptation, Ocular↗

PubMed1986

Light-dependent binding of G-protein to outer segment membranes of toad photoreceptors.

Light-dependent changes in the binding of G-protein were analyzed in outer segment disk membranes obtained from photoreceptors of the toad (Bufo marinus) retina. Isolated, intact retinas, incubated in oxygenated Ringer's solution at 23 +/- 1 degree C, were subjected to various conditions of illumination and then incubated in darkness for specified periods. The retinas were then chilled (0-4 degrees C) and the receptor outer segments (ROS) were isolated. Binding of the alpha- and beta-subunits of G-protein to the ROS membranes was analyzed by quantitating G alpha and G beta extracted from the membranes with hypotonic medium lacking GTP vs. hypotonic medium containing GTP (H and HG extracts, respectively). For retinas illuminated and then immediately chilled for analysis, the extent of G binding (relative abundance of G alpha, beta in the HG extract) increased with the extent of bleaching of the visual pigment. Near-maximal binding was observed after bleaches of greater than or equal to 30%. With an increasing period of incubation in darkness after approximately 70% bleaching, the extent of binding declined gradually to low levels characteristic of unbleached retinas. The period required for half-completion of the decline was approximately 10(3) s. A gradual decline in G binding, from a rapidly developing peak value, was also observed with an increasing period of exposure to intense light. Viewed in the context of previous electrophysiological data, our results indicate that sustained bleaching desensitization of the rods does not depend upon a persisting state of "tight binding" (immobilization) of G-protein by bleached visual pigment.

Adaptation, Physiological↗

PubMed1984

Nucleoside triphosphates and hydrolysis-resistant analogues: effects on PIII responses in the isolated skate retina.

Responses to test flashes were recorded extracellularly from the aspartate-treated, isolated retina of the skate, before and after topical application to the retina of solutions containing GTP, ATP, or certain hydrolysis-resistant analogues. When applied to strongly light-adapted retinas (greater than or equal to 87% bleached), the analogues p(CH2)ppG, p(NH)ppG and p(CH2)ppA induced sustained decreases in threshold of the PIII response. Similar treatment of light-adapted retinas with GTP or ATP also promoted decreases in threshold, but these changes appeared relatively transient. All of the test substances lacked significant activity when applied to dark-adapted (unbleached) retinas. The results are discussed in relation to other studies examining the effects of light on GTP- and ATP-dependent processes within the photoreceptors.

Adaptation, Ocular↗

PubMed1984

Rhodopsin and visual adaptation: analysis of photoreceptor thresholds in the isolated skate retina.

Photoreceptor thresholds in the isolated retina of the skate, determined by extracellular measurement of the photoreceptor potential during periods of light and dark adaptation, were analyzed in relationship to prevailing states of the visual pigment. The starting assumption of the analysis is that relative levels of three forms of the pigment molecule [native rhodopsin (R), a photoactivated intermediate (R*), and bleached pigment (B)] govern (quasi-) stable levels of threshold measured (a) during exposure of the retina to background light of fixed incident intensity (Ib), and (b) after irradiation that bleaches a defined fraction (B) of the rhodopsin. It is shown that experimental data are described well by the equation It/ It0 = (1 - B)-1 X F X (1 + 0(3)B), where F = [1 + 0(1)Ib(1 - B) + 0(2)B]. In this equation, It/ It0 is the relative threshold for detection of a test flash; (1 - B) approximates the relative efficiency of quantum capture; and 0(1) - 0(3) are constants. For values of 0(1) - 0(3) yielding an optimal fit to experimental data, log (It/ It0 ) approximately log F over a broad range of values of Ib and B. It is further shown that the algebraic form of the term F in the above equation is consistent with the predictions of a (steady-state) model for the role of the pigment molecule in photoreceptor adaptation. The model proposes that R* and B desensitize the photoreceptor by acting (in qualitatively similar fashion) to reduce the availability of E, an intracellular substance whose activation supports generation of the flash response. Results of the analysis are discussed in relation to the Dowling- Rushton equation (Dowling, 1960, 1963; Rushton , 1961), and to the results of more recent studies examining light and dark adaptation.

Adaptation, Ocular↗