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H Ripps

Publications and source records attributed to H Ripps.

At least 19 recordsLinked to original sources
PubMed1996

Light-induced acceleration of photoreceptor degeneration in transgenic mice expressing mutant rhodopsin.

PURPOSE: Mutations at various loci on the rhodopsin gene have been shown to cause autosomal dominant retinitis pigmentosa (ADRP). One of the most common is a point mutation (P23H) near the N-terminus of the protein. The authors have studied the effects of light deprivation on the rate of degeneration in pigmented transgenic mice expressing the P23H mutation as well as two additional mutations near the N-terminus of opsin (V20G, P27L). METHODS: Transgenic and normal littermates were reared in darkness or in cyclic light (approximately 7 foot-candle) for periods of 2, 4, or 6 months. Retinal structure and function were evaluated by electroretinography, retinal densitometry, light microscopy, and TUNEL labeling. RESULTS: Retinas of normal animals, whether reared in darkness or in cyclic light, had no structural or functional abnormalities. The rate of photoreceptor degeneration in dark-reared transgenic mice was significantly slower than in transgenic mice raised under cyclic light conditions. Differences between the two groups of animals were evident in the retinal histology, the electroretinographically determined sensitivity to photic stimulation, and the rhodopsin levels in the retina. TUNEL labeling of retinal wholemounts showed that cyclic light-reared animals had a threefold higher incidence of photoreceptor cell death than their dark-reared counterparts; the density of apoptotic cells was greatest in the inferior retina, the region most severely affected in patients with the P23H mutation. In comparison, photoreceptor cell death was more uniformly distributed across the retina in dark-reared transgenic mice. CONCLUSIONS: These findings suggest that light activation of rhodopsin contributes to the severity of the degenerative disease resulting from the P23H opsin mutation, and they raise the possibility that minimizing exposure to light may help to prolong useful vision of patients with this form of retinitis pigmentosa.

Animals↗
PubMed1995

GABA transport and calcium dynamics in horizontal cells from the skate retina.

1. Changes in intracellular calcium concentration [Ca2+]i in response to extracellularly applied gamma-aminobutyric acid (GABA) were studied in isolated horizontal cells from the all-rod skate retina. 2. Calcium measurements were made using fura-2 AM, both with and without whole-cell voltage clamp. Superfusion with GABA, in the absence of voltage clamp, resulted in an increase in [Ca2+]i; the threshold for detection was approximately 50 microM GABA, and a maximal response was elicited by 500 microM GABA. 3. The rise in [Ca2+]i was not mimicked by baclofen nor was it blocked by phaclofen, picrotoxin or bicuculline. However, the GABA-induced [Ca2+]i increase was completely abolished when extracellular sodium was replaced with N-methyl-D-glucamine. 4. With the horizontal cell voltage clamped at -70 mV, GABA evoked a large inward current, but there was no concomitant change in [Ca2+]i. Nifedipine, which blocks L-type voltage-gated Ca2+ channels, suppressed the GABA-induced increase in [Ca2+]i. These findings suggest that the calcium response was initiated by GABA activation of sodium dependent electrogenic transport, and that the resultant depolarization led to the opening of voltage-gated Ca2+ channels, and a rise in [Ca2+]i. 5. The GABA-induced influx of calcium appears not to have been the sole source of the calcium increase. The GABA-induced rise in [Ca2+]i was reduced by dantrolene, indicating that internal Ca2+ stores contributed to the GABA-mediated Ca2+ response. 6. These observations demonstrate that activation of the GABA transporter induces changes in [Ca2+]i which may have important implications for the functional properties of horizontal cells.

Animals↗
PubMed1995

Functional abnormalities in transgenic mice expressing a mutant rhodopsin gene.

PURPOSE: To evaluate the consequences of the expression of a mutant mouse opsin gene on rod- and cone-mediated function. Experimental conditions were chosen to provide a basis of comparison to the results reported for patients with autosomal dominant retinitis pigmentosa (ADRP) in whom the proline at position 23 has been replaced by a histidine (P23H). METHODS: The mutated gene product resulted in three substitutions in the rhodopsin molecule: P23H, glycine for valine at position 20 (V20G), and leucine for proline at position 27 (P27L). Mice positive for the transgene were differentiated from normal littermates by the polymerase chain reaction. Electroretinograms (ERGs) were obtained from anesthetized mice between 1 and 9 months of age. After photically bleaching approximately 18% of the available rhodopsin, the time course of rod dark adaptation was examined by monitoring rod ERG amplitude recovery. Rhodopsin densitometry was used to determine the relative amounts of rhodopsin in the retinae of normal and transgenic mice. RESULTS: ERGs obtained from transgenic mice showed a significant reduction in rod-mediated response amplitude at 1 month of age and a relatively slow progressive decrease thereafter. Cone-mediated ERGs, on the other hand, were nearly normal in amplitude for approximately the first 5 months after birth, but at later ages response amplitudes also underwent a progressive decline. In the normal retina, rod ERG amplitudes returned to prebleach levels within 30 minutes, whereas in transgenic mice response amplitudes did not recover within a 2-hour test period. The age-related decline in rod-mediated electroretinal potentials seen in transgenic mice was paralleled by a concomitant fall in rhodopsin density, and the sensitivity losses obtained electroretinographically could be accounted for solely on the basis of reduced quantal absorption. CONCLUSIONS: The pattern of functional changes seen in the transgenic mice are in good agreement with those reported in patients with ADRP with the P23H mutation in the rhodopsin gene. Particularly noteworthy is the fact that the changes in rhodopsin density and visual sensitivity are associated with a progressive shortening of the rod outer segments; the histologic changes induced by the disease process in patients with ADRP have yet to be determined.

Animals↗
PubMed1994

A novel action of quinine and quinidine on the membrane conductance of neurons from the vertebrate retina.

The cinchona alkaloids quinine and quinidine have been shown to block a broad range of voltage-gated membrane conductances in a variety of excitable tissues. Using the whole-cell version of the patch clamp technique, we examined the effects of these compounds on voltage-dependent currents from horizontal cells dissociated enzymatically from the all-rod retina of the skate. We report here a novel and unexpected action of quinine and quinidine on isolated horizontal cells. In addition to blocking several of the voltage-activated currents of these cells, the introduction of the alkaloids evoked a large outward current when the cells were held at depolarized potentials. Using tail current analysis, the reversal potential of the outward current was close to O mV, and the current was markedly suppressed by extracellularly applied cobalt, acetate, and halothane. Depolarization in the presence of quinine also permitted entry into the cells of extracellularly applied Lucifer yellow (MW = 443 D), whereas a 3-kD fluorescein-dextran complex was excluded. These findings suggest that the large, apparently nonselective conductance induced by quinine and quinidine results from the opening of hemi-gap junctional channels.

Animals↗
PubMed1993

Evidence for hemi-gap junctional channels in isolated horizontal cells of the skate retina.

Prolonged depolarization of isolated, voltage-clamped skate retinal horizontal cells produces an outward current that exhibit a late onset and develops slowly with time. This current, which we refer to as the Q-current, is associated with an increase in membrane conductance, and is present when other voltage-gated conductances have been pharmacologically blocked. The reversal potential for the Q-current, obtained using tail current analysis, was close to 0 mV. The magnitude of the current was greatly reduced by superfusion with 25 mM acetate, and by 4 mM cobalt chloride, 2 mM 1-octanol, and a saturated solution of the general anesthetic halothane. In addition, the low-molecular weight fluorescent dye Lucifer yellow, applied extracellularly, entered the cells during activation of the Q-current, whereas a 3 kD dextran-fluorescein complex did not cross the cell membrane. The effects of divalent cations, the non-specific nature of the ionic current suggested by its reversal potential, the entry of Lucifer yellow, and the ability of acetate, halothane, cobalt, and octanol to block the current lead us to hypothesize that the Q-current results from the opening of hemi-gap junctional channels that mediate electrical coupling between skate horizontal cells.

Animals↗
PubMed1993

Interphotoreceptor retinoid binding protein (IRBP) enhances rhodopsin regeneration in the experimentally detached retina.

Results obtained in a previous study showed that, compared with a normal eyecup preparation, the amount of rhodopsin regenerated and the rate at which it was resynthesized after bleaching were reduced by about 50% when the skate retina was detached from its pigment epithelium (RPE) and replaced immediately on the apical surface of the RPE (Sun and Ripps, 1992). In the present study, these observations have been extended to preparations in which the detachment procedure was performed under fluid in order to dilute the IRBP content of the interphotoreceptor matrix. The goal initially was to determine whether lowering the IRBP concentration of the subretinal space affected the regenerative process. Using fundus reflectometry, it was found that allowing fluid to enter the subretinal space exposed by the detachment procedure caused profound deficits in both the rate and amount of rhodopsin that regenerated after bleaching. Results obtained with SDS-PAGE and immunohistochemistry showed that the molecular weight of the IRBP extracted from the skate retina is similar to that of many other vertebrate species, and that antibodies prepared against mammalian IRBP react with epitopes on skate IRBP within the interphotoreceptor matrix. Accordingly, it was investigated whether it is possible to reverse the detachment-induced anomalies in rhodopsin kinetics by introducing ligand-free IRBP purified from bovine retina to the subretinal space. Again using fundus reflectometry, it was found that instilling 5 microM of a 130 microM IRBP solution between the neural retina and the RPE increased significantly the rate of regeneration, and more than doubled the amount of rhodopsin reformed in darkness.

Adaptation, Ocular↗
PubMed1993

The effects of lowered extracellular sodium on gamma-aminobutyric acid (GABA)-induced currents of Muller (glial) cells of the skate retina.

1. The effects of external sodium on GABA-induced chloride currents were examined with whole-cell voltage-clamp recordings obtained from enzymatically dissociated solitary Muller cells in culture. Our goal was to determine whether a sodium-dependent GABA uptake mechanism influences the GABAa-mediated responses of skate Muller cells. 2. At low concentrations of GABA (0.01 to 0.5 microM), removal of sodium from the external solution resulted in a marked increase in the ligand-gated currents mediated by activation of GABAa receptors. The enhancement by lowered sodium was greatest at hyperpolarizing potentials and decreased progressively as the cell was depolarized. 3. The reversal potential for the GABA-induced response was not significantly altered by the removal of sodium, suggesting that sodium ions did not directly contribute to the GABAa-mediated current. 4. Lowering external sodium had no effect on the currents induced by the GABAa-agonist muscimol, consistent with its much lower affinity for the GABA transport carrier. 5. Application of the GABA uptake blocker nipecotic acid also abolished the effects of lowered sodium. 6. These findings suggest that the effects of lowered external sodium resulted from a decrease in the uptake of GABA into the Muller cells, thus raising the effective concentration of GABA acting upon the GABAa receptors.

Animals↗
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↗
PubMed1993

Ocular ischemia and the effects of allopurinol on functional recovery in the retina of the arterially perfused cat eye.

PURPOSE: This study sought to examine the acute effects of ocular ischemia and reperfusion on retinal function and determine the extent to which recovery during reperfusion is improved by allopurinol (AP), a blocker of xanthine oxidase (XO). The latter is presumed to be a major factor in the formation of free radicals associated with reperfusion of ischemic tissue. METHODS: Electroretinographic (ERG) responses were recorded simultaneously from the two isolated, arterially perfused eyes obtained from the same cat. One eye served as the control and received only the standard perfusate; the other eye was infused with AP before and after a 3 hr period of total ischemia. RESULTS: After the prolonged period of nonperfusion, recovery of the electroretinographic components was incomplete to varying degrees. Maximum b-wave amplitudes recovered only to 17 +/- 5% (mean +/- SEM) of baseline, whereas the a-wave grew to 60 +/- 10% of its baseline value. For both measures, the recovery of electroretinographic amplitude was significantly greater in AP-treated eyes than in the control eyes. CONCLUSION: Generation of free radicals by XO contributes to the retinal damage and loss of function that occurs after a period of ischemia and subsequent reperfusion.

Allopurinol↗
PubMed1992

Rhodopsin regeneration in the normal and in the detached/replaced retina of the skate.

The bleaching and regeneration of rhodopsin in the skate retina was studied by means of fundus reflectometry, both in the normal eyecup preparation and after the retina had been detached and then replaced on the surface of the pigment epithelium (RPE). After bleaching virtually all the rhodopsin in the retinal test area of the normal eyecup, more than 90% of the photopigment was reformed after about 2 hr in darkness; over most of this time course, rhodopsin density rose linearly at a rate of 0.875% min-1 with a half-time of 55 min. Detaching the retina from its pigment epithelium resulted in a number of abnormalities, both structural and functional. Histological examination of the detached/replaced (D/R) retina showed striking alterations in the structural integrity of the RPE cells at their interface with the neural retina. The cells appeared vacuolated and misshapen, and the apical processes of the RPE, which normally ensheath the receptor outer segments, were shredded and free of their association with the visual cells. These morphological changes, as well as dilution of the IRBP content of the subretinal space caused by separation of the tissues, appear to be the main factors contributing to the functional abnormalities in rhodopsin kinetics. But despite these abnormalities and the persistent detachment, the rate of regeneration and the amount of rhodopsin reformed after bleaching were reduced by less than 50% of their normal values. The fact that a significant fraction of the bleached rhodopsin was regenerated under these conditions indicates that 11-cis retinal formed in the RPE was able to traverse a much greater than normal subretinal space to reach the opsin-bearing photoreceptor membranes.

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↗
PubMed1992

Receptive field properties of rod-driven horizontal cells in the skate retina.

The large receptive fields of retinal horizontal cells result primarily from extensive intercellular coupling via gap (electrical) junctions; thus, the extent of the receptive field provides an index of the degree to which the cells are electrically coupled. For rod-driven horizontal cells in the dark-adapted skate retina, a space constant of 1.18 +/- 0.15 mm (SD) was obtained from measurements with a moving slit stimulus, and a comparable value (1.43 +/- 0.55 mm) was obtained with variation in spot diameter. These values, and the extensive spread of a fluorescent dye (Lucifer Yellow) from the site of injection to neighboring cells, indicate that the horizontal cells of the all-rod retina of skate are well coupled electrically. Neither the receptive field properties nor the gap-junctional features of skate horizontal cells were influenced by the adaptive state of the retina: (a) the receptive field organization was unaffected by light adaptation, (b) similar dye coupling was seen in both dark- and light-adapted retinae, and (c) no significant differences were found in the gap-junctional particle densities measured in dark- and light-adapted retinas, i.e., 3,184 +/- 286/microns 2 (n = 8) and 3,073 +/- 494/microns 2 (n = 11), respectively. Moreover, the receptive fields of skate horizontal cells were not altered by either dopamine, glycine, GABA, or the GABAA receptor antagonists bicuculline and picrotoxin. We conclude that the rod-driven horizontal cells of the skate retina are tightly coupled to one another, and that the coupling is not affected by photic and pharmacological conditions that are known to modulate intercellular coupling between cone-driven horizontal cells in other species.

Animals↗
PubMed1991

Antiretinal antibodies in serum of patients with age-related macular degeneration.

Antibodies, immunoreactive with normal human retinal proteins, were detected by Western immunoblot analysis in the sera of 30 patients with age-related macular degeneration (AMD). Sera from 14 of these patients demonstrated positive binding predominantly to a doublet protein of molecular weight between 58 and 62 kD. The sera from the remaining 16 patients and from 12 control subjects reacted either weakly or not at all with the doublet protein. No correlation was found with any specific type of AMD. The serum antibodies also immunocrossreacted with the same proteins from isolated photoreceptor outer segments; this was confirmed by indirect immunofluorescence on intact retinas. The crossreactivity of the serum antibodies with a protein of Mr 58 to 62 kD, the lower band present in the bovine purified neurofilament-68 kD preparation, suggests strongly that this protein may be a component of the neuronal cytoskeleton. However, it is not clear whether these autoantibodies play a direct role in the etiology of AMD or represent a nonspecific response to retinal damage.

Aged↗
PubMed1991

Ultrastructural and electrophysiological changes associated with K(+)-evoked release of neurotransmitter at the synaptic terminals of skate photoreceptors.

Bathing the skate retina in a Ringer solution containing a high concentration (100 mM) of potassium ions depolarized the visual cells, depleted the receptor terminals of synaptic vesicles, and suppressed completely the b-wave of the ERG and the intracellularly recorded response of horizontal cells (the S-potential). The depletion of synaptic vesicles was accompanied by a large increase in the extent of the plasma membrane resulting in distortion of the normal terminal profile, i.e. distension of the basal surface and elaborate infolding of protoplasmic extensions. Morphometric analysis showed that despite the changes in vesicle content and terminal structure, the combined linear extent of the vesicular and plasma membranes was unchanged from control (superfusion with normal Ringer solution); the increase in plasma membrane was equivalent to the observed loss of vesicular membrane. When returned to a normal Ringer solution, the terminals rapidly began to reform, and in about 10 min they were morphologically indistinguishable from receptor terminals seen in control preparations. After 30 min in the normal Ringer solution, the amount of membrane associated with the vesicles and the plasma membrane had reverted to control values, and once again the total membrane estimated morphometrically remained essentially the same. Thus, there is an efficient mechanism at the photoreceptor terminal for the recycling of vesicle membrane following exocytosis. The K(+)-induced depletion of synaptic vesicles was paralleled by a precipitous loss of responsivity in both the b-wave of the ERG and the S-potential of the horizontal cells. However, after 30-min exposure to the high K+ and a return to normal Ringer solution, the recovery of electrophysiological activity followed a much slower time course from that associated with the structural changes; 60 min or longer were required for the potentials to exhibit maximum response amplitudes. It appears that the rate-limiting step in restoring normal synaptic function following massive depletion of vesicular stores is transmitter resynthesis and vesicle loading rather than vesicle recycling.

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

Structural features and adaptive properties of photoreceptors in the skate retina.

In the duplex (rod/cone) retinae of most vertebrates, electrical responses associated with nocturnal vision can be recorded in relative isolation only over a limited intensity range before there is encroachment by signals from the cone system; e.g., at mesopic levels of illumination, the rods begin to saturate, and the cone mechanism is brought into play. On the other hand, the retinae of some sub-species of skate appear to contain only one class of photoreceptors, namely rods. Nevertheless, skate photoreceptors, as well as other retinal neurons, are able to respond to incremental stimuli presented on background fields so intense that 90% or more of the available rhodopsin is bleached during the exposure. These and other findings raise some doubts as to whether the skate has, in fact, an all-rod retina. In this paper, we present a body of evidence--based on the results of photochemical, anatomical, and electrophysiological studies--to support the view that only a single class of photoreceptor subserves vision in the skate retina. In addition, recent findings will be described that: a) demonstrate how the visual cells transform from sluggishly responding rods to brisk, cone-like elements, and b) may provide some insight into the functional significance of this unusual adaptive property.

Adaptation, Ocular↗