Biomedical subjects

N J Mangini

Publications and source records attributed to N J Mangini.

At least 19 recordsLinked to original sources

PubMed2002

Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium.

Retinal pigment epithelium (RPE) possesses regulated chloride channels that are crucial for transepithelial fluid and ion transport. At present, little is known about the molecular nature of chloride channels in human adult RPE (haRPE) or the effects of oxidative stress on membrane conductance properties. In the present study, we assessed ClC channel and cystic fibrosis transmembrane conductance regulator (CFTR) expression and membrane chloride conductance properties in haRPE cells. ClC-5, ClC-3, ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, and protein expression was detected with Western blot analysis and immunofluorescence microscopy. Whole cell recordings of primary cultures of haRPE showed an outwardly rectifying chloride current that was inhibited by the oxidant H(2)O(2). The inhibitory effects of H(2)O(2) were reduced in cultured human RPE cells that were incubated with precursors of glutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate a possible role for ClC channels in haRPE cells and suggest possible redox modulation of human RPE chloride conductances.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Creatine kinase in human retinal pigment epithelium.

Retinal pigment epithelial ion transport activity, and consequent ATP consumption vary significantly as a function of photoreceptor activity. In a variety of cell types, ATP levels are maintained during high-energy usage by phosphocreatine hydrolysis, catalysed by the enzyme creatine kinase. The present work was designed to assess the importance of creatine kinase in retinal pigment epithelial cell metabolism. To this end, activity measurements, non-denaturing gel electrophoresis, Western blot analysis and immunohistochemistry were used to characterize creatine kinase in retinal pigment epithelium. Total creatine kinase activity in the retinal pigment epithelium is approximately 0.05 micromol ATP mg protein(-1) min(-1). The bulk of this activity was mediated by the B-CK isoform. However, by immunoblotting, non-denaturing gel electrophoresis and immunohistochemistry, the presence of the M-CK isoform of creatine kinase was also detected. The M-CK isoform was plasma membrane associated and predominately localized to the apical surface. Creatine kinase in the retinal pigment epithelium could function in a spatial energy shuttle that helps to sustain apical plasma membrane ion transport activity.

Blotting, Western↗

PubMed1999

Transthyretin localization in cultured and native human retinal pigment epithelium.

The aim of this study was to determine transthyretin subcellular localization in cultured and native human retinal pigment epithelium. Monoclonal and polyclonal antibodies directed against human plasma transthyretin were used to detect transthyretin-specific immunoreactivity in cultured human retinal pigment epithelium. At the light microscopic level, transthyretin-specific immunoreactivity was observed throughout the cytosol with intense perinuclear staining. Nuclear staining was faint, but detectable. Both monoclonal and polyclonal antibodies exhibited similar staining patterns. An electron microscopic immunogold labeling protocol detected transthyretin-specific immunoreactivity in cultured and native human retinal pigment epithelium. Transthyretin-specific immunogold labeling within mitochondrial, apical, basal, nuclear and cytosolic subcellular compartments was quantitated and statistically analyzed. The pattern of transthyretin labeling was similar for each antibody, and comparable between cultured and native human retinal pigment epithelium. Transthyretin labeling was observed in mitochondrial and nuclear compartments, and in close apposition to both apical and basal membranes. Transthyretin labeling density was highest in the mitochondrial compartment and was significantly greater than labeling in all other compartments. Detection of transthyretin labeling in mitochondrial and nuclear compartments suggests an intracellular role for transthyretin in human retinal pigment epithelium, possibly as a cytoplasmic carrier protein for thyroxine.

Aged↗

PubMed1998

Immunohistochemical localization of Na+/Ca2+ exchanger in human retina and retinal pigment epithelium.

BACKGROUND: Impaired calcium (Ca2+) metabolism has been implicated in the pathogenesis of various ocular diseases, suggesting that regulation of Ca2+ homeostasis in the retina is of major significance for normal function. There are known families of transport proteins that can catalyze net Ca2+ efflux across the plasma membrane, one of which is the Na+/Ca2+ exchanger. Using immunohistochemistry, we have investigated the human retina and retinal pigment epithelium (RPE) for the presence and distribution of the so-called cardiac-type Na+/Ca2+ exchanger. METHODS: Paraffin sections of ten eyes enucleated for various disease processes were incubated according to the ABC method with a polyclonal antibody (pi) produced against the canine cardiac sarcolemmal Na+/Ca2+ exchanger. The reaction was visualized with aminoethylcarbazole. RESULTS: There was a positive reaction with anti-Na+/Ca2+ exchanger in the retina and RPE in all eyes, but the labeling varied among the different specimens. In neural retina, staining was most intense in Mueller cells, in cells of the inner nuclear layer, and in cone inner segments. Immunoreactivity was less pronounced in ganglion cells, nerve fibers, the outer nuclear layer and in rod inner segments. Outer segments appeared mostly negative. In the RPE, positive staining was present but the intensity of staining varied both within and among the specimens. Reactive RPE cells revealed the most intense labeling. CONCLUSION: Na+/Ca2+ exchanger of the cardiac type is present in human retina and RPE. The variation in immunoreactivity among the different specimens may reflect the different diseases of these eyes and their different metabolic states. A specific relation between certain diseases and malfunction of the Na+/Ca2+ exchanger could have a major impact on therapeutic regimens.

Adolescent↗

PubMed1997

Unscheduled DNA replication precedes apoptosis of photoreceptors expressing SV40 T antigen.

Expression of simian virus 40 T antigen (Tag) in the rod photoreceptors of transgenic mice leads to cell death that is completed by the end of the third week of postnatal development. To understand the mechanistic link between Tag expression and the death of the expressing photoreceptors, cell cycle activity was followed in a transgenic mouse family that expresses Tag directed by the mouse opsin promoter. Tag-expressing photoreceptors also expressed rhodopsin suggesting that these cells were differentiated. The presence of Tag in the photoreceptors induced the expression of both proliferating cell nuclear antigen (PCNA) and thymidine kinase (TK). The abnormally high levels of PCNA and TK continued until the complete disappearance of the cells expressing Tag. Photoreceptor cell death was also associated with continued DNA synthesis that ceased shortly after postnatal day 16. The specific loss of the rod photoreceptors that re-entered the cell cycle accounted entirely for the loss of photoreceptors from the outer nuclear layer. The antiproliferative nature of the mature retina is directly involved in the apoptotic death of photoreceptors expressing Tag.

Animals↗

PubMed1997

Sodium-calcium exchanger in cultured human retinal pigment epithelium.

Regulation of intracellular free Ca2+ concentration ([Ca2+]i) by an Na+/Ca2+ exchanger was studied in cultures of human retinal pigment epithelial cells using Ca(2+)-indicator dyes (fura-2 and fluo-3) and digital fluorescence imaging. Mean resting [Ca2+]i of cultured RPE in a control Ringer solution was 189 +/- 16 nM. Replacing extracellular Na+ with N-methyl-D-glucamine elicited a two-fold rise in [Ca2+]i; the magnitude of the [Na+]o-free-induced rise in [Ca2+]i varied as a function of extracellular [Ca2+]. The [Na+]o-free response was not significantly affected by the Ca2+ channel blocker nifedipine, or by pretreatment with thapsigargin which depletes intracellular Ca2+ stores. By contrast, the [Na+]o-free-induced rise in [Ca2+]i was significantly reduced by CBDMB, an amiloride derivative that is highly selective for Na+/Ca2+ exchange inhibition. These findings indicate that removal of extracellular Na+ promotes net [Ca2+]i gain via Na+/Ca2+ exchange. Western and Northern blot analyses, respectively, confirmed the presence of Na+/Ca2+ exchanger protein and mRNA in cultures of human RPE. Specifically, Western blot analysis of whole cell lysates of cultured RPE using a polyclonal antibody made against the canine cardiac exchanger identified a major band at approximately 126 kD. Northern blot analysis of total human RPE RNA using a restriction fragment cRNA probe coding for the canine cardiac Na+/Ca2+ exchanger showed that the major exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-type Na+/Ca2+ exchanger in cultures of human RPE.

Adult↗

PubMed1997

Immunolocalization of ubiquitin and related enzymes in human retina and retinal pigment epithelium.

PURPOSE: To examine the localization of ubiquitin (Ub) and related enzymes in human retina with emphasis on the retinal pigment epithelium (RPE)-Bruch's membrane complex. METHODS: Thirty human eyes enucleated for various disease processes were examined. Immunohistochemistry was performed on paraffin sections using antibodies against Ub, Ub-conjugating enzyme (E2), Ub carboxyl-terminal hydrolase (PGP 9.5), and, for comparison, arrestin (Arr). Immunoreactivity (IR) was tested using the avidin-biotin method. RESULTS: Ub was present throughout retina but was particularly prominent in ganglion cells and RPE. Most intriguing was the presence of Ub IR in age-related, sub-RPE deposits such as drusen and basal laminar deposits (BLD). RPE immunolabeling was more intense in older tissue, but otherwise no pattern of Ub IR could be linked to specific diseases. E2 IR colocalized with Ub, with one exception; E2 IR was not found in drusen or BLD. PGP 9.5 IR was intense in nerve fibers, ganglion cells, and the inner nuclear and plexiform layers. RPE staining was faint and patchy; sub-RPE deposits were not labeled. Arr IR was present in photoreceptors but not within or beneath RPE cells. CONCLUSION: The ubiquitination process is important in human retina and particularly in ganglion cells. Ub-related processes are also active in RPE and may be involved in the degradation and disposal of proteins from these cells. The presence of Ub in sub-RPE deposits--without related Ub-processing enzymes--raises the possibility that certain proteins become ubiquitinated within RPE but that further degradation of the Ub-protein complexes does not occur.

Adult↗

PubMed1996

Plasma membrane calcium-ATPase in cultured human retinal pigment epithelium.

The present work demonstrates the presence of plasma membrane (Ca(2+) + Mg2+)-ATPase (PMCA) activity in cultured human retinal pigment epithelium (HRPE). Whole-cell HRPE homogenates exhibited Ca(2+)-stimulated ATPase activity that was inhibited with high affinity (IC50 = 60 nM) by eosin, a potent inhibitor of the erythrocyte PMCA. This activity was not inhibited by thapsigargin, a selective inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA). A PMCA-specific monoclonal antibody exhibited staining in attached HRPE monolayers. By Western blot analysis of SDS-PAGE separations of whole cell lysates of HRPE, this antibody identified a single band at approximately 145 kD. Finally, a Ca(2+)-dependent, La(3+)-augmented phosphoprotein, which comigrated with anti-PMCA immunoreactivity, was also detected. Taken together, these results show that cultured HRPE express PMCA activity.

Blotting, Western↗

PubMed1996

Immunodetection of an arrestin-like protein in human retinal pigment epithelium.

The goal of this study was to determine if an arrestin/S-antigen-like protein is produced by human retinal pigment epithelial (HRPE) cells maintained in tissue culture. Arrestin immunoreactivity was examined in fixed, monolayer cultures of HRPE and on immunoblots of SDS-PAGE separations of whole cell lysates of HRPE using five monoclonal antibodies (mAbs A2G5, A9C6, 3C4.2, 3D1.2 and 5c6.47) that bind to different epitopes in bovine retinal S-antigen. Monolayers of HRPE cells showed immunoreactivity with four of the mAbs though the relative staining intensity varied among mAbs and donors. For example, mAb A2G5 which historically shows very limited crossreactivity among species, reacted strongly with cells from one donor, moderately with cells from a second donor and only weakly with other donor cultures examined. mAb 3D1.2 showed no reactivity with HRPE cells. Immunoblots of SDS-PAGE separations of whole cell lysates of HRPE established from ten different donors confirmed the presence of an arrestin-related polypeptide that comigrated with retinal arrestin. These results demonstrate the presence of an arrestin-like protein in HRPE cells which have been maintained in tissue culture. Though the function of this arrestin homologue in HRPE is not yet established, it could play a role in the downregulation of receptor and/or transport protein activity.

Aged↗

PubMed1995

Reduced light-dependent phosphorylation of an analog visual pigment containing 9-demethylretinal as its chromophore.

9-Demethyl rhodopsin (9dR), an analog of vertebrate rhodopsin, consists of opsin and a covalently attached chromophore of 11-cis 9-demethylretinal. Electrophysiological evidence that photoactivated 9dR (9dR*) undergoes abnormally slow deactivation in salamander rods (Corson, D. W., Cornwall, M. C., and Pepperberg, D. R. (1994) Visual Neurosci. 11, 91-98) raises the possibility that opsin phosphorylation, a reaction involved in visual pigment deactivation, operates abnormally on 9dR*. This possibility was tested by measuring the light-dependent phosphorylation of 9dR in preparations obtained from bovine rod outer segments. Outer segment membranes containing 9dR or regenerated rhodopsin were flash-illuminated in the presence of [gamma-32P]ATP and rhodopsin kinase, further incubated in darkness, and then analyzed for opsin-bound [32P]Pi. [32P]Pi incorporation by 9dR* increased with both incubation period and bleaching extent but, under all conditions tested, was less than that measured in rhodopsin controls. Results obtained with 30-s incubation periods indicated that the maximal initial rate of incorporation by 9dR* is about 25% of that by photoactivated rhodopsin. The results imply that the low incorporation of Pi by 9dR* results from a reduced rate of phosphorylation by rhodopsin kinase and are consistent with the prolonged lifetime of 9dR* determined electrophysiologically.

Animals↗

PubMed1995

Anti-arrestin immunoreactivity in the human retina: difference between light- and dark-adaptation.

Differences in arrestin (Arr) immunolocalization between light(LA)- and dark(DA)-adapted retinae have been described in various species. We have for the first time studied 5 LA and 5 DA human retinae from surgically enucleated eyes, each group comprising 1 exenteration specimen and globes with malignant melanoma or 2 degrees glaucoma. To examine the distribution of Arr, immunohistochemistry was performed on paraffin sections using three different antibodies to Arr: a48k, 3D1.2, and 5c6.47. Immunoreactivity (IR) was tested using the avidin-biotin method, and results were visualized with diaminobenzidine. With both a48k and 5c6.47, labeling was most intense in the photoreceptor layer. When comparing LA and DA retinae, IR of photoreceptor outer segments (OS) was clearly different with very little IR in OS of DA but distinct positivity in OS of LA retina. This was most obvious in melanoma eyes where retinal morphology was well-preserved while in glaucomatous eyes with retinal degeneration this pattern was less apparent. 3D1.2 did not react in any of the specimens. Although there was some variation within each group, we demonstrated a distinct difference in anti-Arr IR between LA and DA specimens. Thus Arr-IR might now be used as a promising tool to further study retinal diseases on human surgical specimens involving photoreceptor degeneration.

Adaptation, Ocular↗

PubMed1993

Rods and cones contain antigenically distinctive S-antigens.

PURPOSE: S-antigen (48 kDa protein or arrestin) is known to be present in rod photoreceptors. Its localization in cones is less clear with several conflicting reports among various species examined. METHODS: This study employed three different anti-S-antigen antibodies (a48K, a polyclonal antiserum and two monoclonal antibodies, MAb A9-C6 and MAb 5c6.47) and examined their localization in rods and cones of human and cat retinas. To identify the respective cone types, an enzyme histochemical technique for carbonic anhydrase (CA) was employed to distinguish blue cones (CA-negative) from red or green cones (CA-positive). S-antigen localization was then examined by immunocytochemical staining of adjacent sections. RESULTS: In human retinas, a similar labeling pattern was seen with both a48K and MAb A9-C6, i.e., the rods and blue-sensitive cones were strongly positive, whereas the red- or green-sensitive cones showed little immunoreactivity. All human photoreceptors showed reactivity to MAb 5c6.47. In the cat retina, only CA-positive cones could be found. As in the human retina, both rods and cones of the cat were positive for MAb 5c6.47. A difference from the labeling pattern in human retina was noted for the other S-antigen antibodies; a48K labeled rods and all of the cones, whereas MAb A9-C6 reacted strongly with the rods but showed no cone staining. CONCLUSIONS: These results suggest that both rods and cones contain S-antigen but that they are antigenically distinctive.

Amino Acid Sequence↗

PubMed1991

Immunolocalization of arrestin (S-antigen) in rods of pearl mutant and wild-type mice.

The pearl mutant mouse is hypopigmented and exhibits a significantly elevated dark-adapted (DA) threshold in comparison to the congenic wild-type mouse. The primary cause of the elevated DA threshold is not known. The subcellular immunolocalization of arrestin/S-antigen reflects the state of adaptation of rod photoreceptors. In this study, quantitative immunoelectron microscopy was used to examine the subcellular distribution of arrestin in wild-type and pearl rods as a function of light exposure. The goal was to determine whether arrestin distribution within rods of pearl and wild-type mice responds to background luminance in a comparable manner. The level of arrestin immunolabeling in DA (unilluminated) rods of pearl retinas was indistinguishable from that measured in wild-type rods. By contrast, arrestin immunolabeling in light-adapted (LA) pearl rod outer segments (ROS) was significantly greater than in wild-type LA ROS. Relative to DA ROS, arrestin labeling density increased 1.6 fold in wild-type ROS following light adaptation, as compared to a 4 fold increase in pearl ROS. These data suggest that although arrestin levels in DA pearl rods are indistinguishable from that of DA wild-type rods, net changes in arrestin immunolocalization in response to light exposure reflect the effects of the pearl mutation at the level of the rod outer segment. The possible implication of this finding is discussed in view of the proposed role of arrestin in the down-regulation of the enzymatic cascade 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

Retinopretectal and accessory optic projections of normal mice and the OKN-defective mutant mice beige, beige-J, and pearl.

Retinal projections to the pretectal and terminal accessory optic nuclei were studied in normal wild-type mice and mutant mice with abnormal optokinetic nystagmus (OKN, Mangini, Vanable, Williams, and Pinto: J. Comp. Neurol. 241:191-209, '85). The mutants used were pearl, which exhibits an inverted OKN in response to stimulation of only the temporal retina, and beige and beige-J, which show inverted OKN in response to stimulation of only the temporal retina and, in addition, exhibit eye movements with a vertical component in response to horizontally moving, full-field stimuli. These projections were studied following intraocular injections of 3H-proline or horseradish peroxidase (HRP) with, respectively, light microscopic autoradiography or HRP histochemistry. In wild-type mice, strong contralateral retinal projections covered the entire nucleus of the optic tract, the anterior and posterior divisions of the olivary pretectal nucleus, and the posterior pretectal nucleus. Similar heavy contralateral projections were distributed over the dorsal and medial terminal nuclei of the accessory optic system. Also, terminals sparsely covered the entire neuropil of the contralateral lateral terminal nucleus in some but not all wild-type mice. The most prominent accessory optic input was to the medial terminal nucleus and was provided by the inferior fasciculus of the accessory optic tract. A typical mammalian superior fasciculus of the accessory optic system with anterior, middle, and posterior components was present. Ipsilateral label was found in anterior and posterior olivary pretectal nuclei in all of the wild-type animals, but was found inconsistently in the ipsilateral terminal accessory optic nuclei. The pattern of contralateral retinal projection to the nucleus of the optic tract and posterior pretectal nucleus in mutants was indistinguishable from that seen in the normal wild-type mice. However, retinal inputs to the ipsilateral anterior and posterior olivary pretectal nuclei were significantly reduced in pearl mutants and were exceedingly sparse in the beige and beige-J mutant mice, while the contralateral inputs to these nuclei were increased in a complementary fashion in the mutants. The labeling of the accessory optic input to the contralateral dorsal terminal nucleus appeared to be substantially reduced in all of the mutant mice. The size of the principal accessory optic fascicle, the inferior fasciculus, was significantly smaller in beige, beige-J, and pearl mice; this reduction was greater in the beige and beige-J than in the pearl mice.(ABSTRACT TRUNCATED AT 400 WORDS)

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↗