Nondestructive Measurement of Retinal Glucose Transport and Consumption In Vivo Using NMR Spectroscopy

Abstract: The cellular events underlying various retinopathies are poorly understood but likely involve perturbation of retinal glucose metabolism. Current methods for assessing this metabolism are destructive, thus limiting longitudinal studies. We hypothesize that following an intravitreous injection, the clearance rate of a glucose analogue will be a nondestructive index of retinal glucose transport and metabolism in vivo. First, radiolabeled glucose analogues were injected into the vitreous. After 40 min, the dominant clearance path was posterior via the retina and was consistent with a facilitated transport mechanism. Next, either [6,6-²H₂]glucose or 3-deoxy-3-fluoro- d -glucose was injected into the vitreous of rabbit eyes, and the clearance rate of each analogue was determined over 40 min using, respectively, ²H or ¹⁹F NMR. These rates were interpreted as a function of the retinal glucose transport and consumption. From the NMR data, the rate of retinal glucose consumption was ∼16 times slower than the transport of glucose. These data demonstrate that NMR measurements of glucose analogue clearance rate from the vitreous can provide a nondestructive index of retinal glucose transport and consumption in vivo.     

Probing a polar cluster in the retinal binding pocket of bacteriorhodopsin by a chemical design approach

Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C(13) of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoproteins with all-trans-retinal and its 20-methyl derivative (hereafter, 13-ethyl retinal). Biophysical characterization indicates that recovering the steric interaction between the residue 90 and retinal, eases the accommodation of the chromophore, however it is not enough for a complete phenotype rescue. The characterization of these chemically engineered chromoproteins provides further insight into the role of the hydrogen bond network and the steric interactions involving the retinal binding pocket in bacteriorhodopsin and other microbial sensory rhodopsins.     

All-trans/13-cis isomerization of retinal is required for phototaxis signaling by sensory rhodopsins in Halobacterium halobium.

An analogue of all-trans retinal in which all-trans/13-cis isomerization is blocked by a carbon bridge from C12 to C14 was incorporated into the apoproteins of sensory rhodopsin I (SR-I) and sensory rhodopsin II (SR-II, also called phoborhodopsin) in retinal-deficient Halobacterium halobium membranes. The "all-trans-locked" retinal analogue forms SR-I and SR-II analogue pigments with similar absorption spectra as the native pigments. Blocking isomerization prevents the formation of the long-lived intermediate of the SR-I photocycle (S373) and those of the SR-II photocycle (S-II360 and S-II530). A computerized cell tracking and motion analysis system capable of detecting 2% of native pigment activity was used for assessing motility behavior. Introduction of the locked analogue into SR-I or SR-II apoprotein in vivo did not restore phototactic responses through any of the three known photosensory systems (SR-I attractant, SR-I repellent, or SR-II repellent). We conclude that unlike the phototaxis receptor of Chlamydomonas reinhardtii, which has been reported to mediate physiological responses without specific double-bond isomerization of its retinal chromophore (Foster et al., 1989), all-trans/13-cis isomerization is essential for SR-I and SR-II phototaxis signaling.     

Picosecond kinetic absorption and fluorescence studies of bovine rhodopsin with a fixed 11-ene

A synthetic retinal having a fixed 11-cis geometry has been used to prepare a nonbleachable analogue of bovine rhodopsin. Marked differences in the picosecond absorption and fluorescence behavior of this analogue at room temperature, compared with that of natural rhodopsin, were observed. This not only indicates that the 11-cis to trans isomerization of the retinal moiety is the crucial primary event in the photolysis of rhodopsin, but also it establishes that this isomerization must occur on the picosecond time scale or faster.     

Effect of variation of retinal polyene side-chain length on formation and function of bacteriorhodopsin analogue pigments

The effect of the length of the retinal polyene side chain on bacterioopsin pigment formation and function has been investigated with two series of synthetic retinal analogues. Cyclohexyl derivatives with polyene chains one carbon longer and one or more carbons shorter than retinal and linear polyenes with no ring have been synthesized and characterized. Compounds of six carbons or less in the polyene chain form pigments very poorly or not at all with bacterioopsin. Compounds containing at least seven carbons in the chain are found to form reasonably stable bacterioopsin pigments that show a small shift in absorbance on irradiation. However, photocycling and proton photorelease are not detected. The analogue with nine carbons in the polyene chain (one less than retinal) forms a stable pigment with an M-type intermediate but demonstrates reduced amounts of photocycling and light-activated proton release. The analogue with a polyene chain identical with that of retinal, but containing no ring, forms a pigment that shows both an efficient light-activated proton photocycle and release. The pigment containing the chromophore with the polyene chain one carbon longer than retinal is likewise fully active. We thus conclude that the length of the polyene chain must be at least 9 carbons for the formation of a stable pigment that photocycles and must be 10 carbons for both the photocycle and light-activated proton release to have a high quantum efficiency.     

Depletion of retinal taurine by treatment with guanidinoethyl sulfonate

Treatment of rats with guanidinoethyl sulfonate, an analogue of taurine, led to a significant decrease in concentration of retinal taurine. These findings suggest that transport of taurine across the blood-retinal barrier is required to maintain retinal taurine levels.     

Halorhodopsin and sensory rhodopsin contain a C6-C7 s-trans retinal chromophore.

Halorhodopsin (HR) and sensory rhodopsin (SR) have been regenerated with retinal analogues that are covalently locked in the 6-s-cis or 6-s-trans conformations. Both pigments regenerate more completely with the locked 6-s-trans retinal and produce analogue pigments with absorption maxima (577 nm for HR and 592 nm for SR) nearly identical to those of the native pigments (577 and 587 nm). This indicates that HR and SR bind retinal in the 6-s-trans conformation. The opsin shift for the locked 6-s-trans analogue in HR is 1,200 cm-1 less than that for the native chromophore (5,400 cm-1). The opsin shift for the 6-s-trans analogue in SR is 1,100 cm-1 less than that for the native retinal (5,700 cm-1). This demonstrates that approximately 20% of the opsin shift in these pigments arises from a protein-induced change in the chromophore conformation from twisted 6-s-cis in solution to planar 6-s-trans in the protein. The reduced opsin shift observed for the locked 6-s-cis analogue pigments compared with the locked 6-s-trans pigments may be due to a positive electrostatic perturbation near C7.     

2-Amino-4-Phosphonobutyric Acid Exerts a Light-Dependent Effect on Post-Gabaculine Levels of Retinal γ-Aminobutyric Acid (GABA): Evidence that ON Synaptic Pathways Regulate Retinal GABAergic Transmission

Abstract: The effects of light, 2-amino-4-phosphonobutyric acid (APB), and kainic acid on rat retinal γ-aminobutyric acid (GABA)-ergic transmission were studied by measuring levels of retinal GABA following subcutaneous injection of gabaculine, an irreversible inhibitor of GABA-transaminase. Post-gabaculine levels of retinal GABA in light-exposed rats were significantly greater than those in rats held in darkness. The synaptic mechanism of this effect of light was examined by measuring post-gabaculine levels of retinal GABA in rats placed into either lighted or darkened conditions after receiving unilateral intravitreal injections of APB, a glutamate analogue that selectively decreases the activity of ON synaptic pathways in the retina. APB attenuated the post-gabaculine accumulation of GABA in rats held in the light, but not in those placed into darkness. Furthermore, the light-dependent increment in post-gabacu line accumulation of retinal GABA was entirely APB sensitive, and the effect of APB was entirely light dependent. In contrast to APB, kainic acid stimulated the post-gabaculine accumulation of retinal GABA in vivo. Our findings suggest that APB and kainic acid influence GABAergic transmission at different sites in the retina and that some retinal GABAergic neurons are either ON or ON-OFF amacrine cells.     

The in vivo cleavage of carotenoids into retinoids in Chlamydomonas reinhardtii

A variety of carotenoids have been incorporated in vivo intoa carotenoid mutant of the unicellular alga, Chlamydomonas reinhardtii.The mutant can synthesize retinal from exogenous carotenoids.In this way the first step in biosynthesis of retinoids, namelyhow ß-carotene is converted into retinal, has beenstudied. Since retinal forms with opsin the photoreceptor forphototaxis in Chlamydomonas, the action-spectral peaks of thephototaxis restored by carotenoid incorporation were used topredict the products formed by the enzyme that cleaves thesecarotenoids. The data suggest that the physiologically relevantcleavage of ß-carotene into retinal is central ratherthan excentric. When apocarotenoids are substrates, the enzymetargets the double bond located a constant distance away fromthe carbonyl group on the acyclic end and, consequently, retinalis not produced. Interestingly, dehydro-analogues containinga triple bond are preferentially cleaved and oxidized at thetriple bond relative to the corresponding analogue with onlydouble bonds. Key words: Chlamydomonas reinhardtii, Chlorophyceae, retinoid biosynthesis, carotenoid cleavage, action spectrum     

Synthetic pigment analogues of the purple membrane protein.

Nonphysiological analogues of retinal have been shown to form pigments in reactions with the apoprotein of the purple membrane of Halobacterium halobium. Both the all-trans and 13-cis isomers of a retinal analogue, having an elongated chain with an extra double bond, formed pigments. Unlike the native all-trans and 13-cis retinal1-based pigments, the new pigments were not interconvertible with each other and were unstable against hydroxylamine. When incorporated into phospholipid vesicles, they showed no proton pumping activity upon illumination. The ability of the extended-length retinal to form pigments contrasts with its nonreactivity with opsin (apoprotein of rhodopsin), suggesting a less stringent binding site for the purple membrane chromophore. All-trans retinal2 also combined with bleached purple membrane to form a blue pigment absorbing at ca. 590 nm. Like the native purple membrane, the blu membrane showed proton pumping activity upon illumination in phospholipid vesicles.     

Constraints of the 9-methyl group binding pocket of the rhodopsin chromophore probed by 9-halogeno substitution

Sterical constraints of the 9-methyl-binding pocket of the rhodopsin chromophore are probed using retinal analogues carrying substituents of increasing size at the 9 position (H, F, Cl, Br, CH(3), and I). The corresponding 11-Z retinals were employed to investigate formation of photosensitive pigment, and the primary photoproduct was identified by Fourier transform infrared difference spectroscopy. In addition, any effects of cumulative strain were studied by introduction of the 9-Z configuration and/or the alpha-retinal ring structure.Our results show that the 9-F analogue still can escape from the 9-methyl-binding pocket and that its photochemistry behaves very similar to the 9-demethyl analogue. The 9-Cl and 9-Br analogues behave very similar to the native 9-methyl pigments, but the 9-I retinal does not fit very well and shows poor pigment formation. This puts an upper limit on the radial dimension of the 9-methyl pocket at 0.45-0.50 nm. Introduction of the alpha-retinal ring constraint in the 11-Z series results in cumulative strain, because the 9-I and 9-Br derivatives cannot bind to generate a photopigment. The 9-Z configuration can partially compensate for the additional alpha-retinal strain. The corresponding 9-Br analogue does form a photopigment, and the other derivatives give increased photopigment yields compared to the corresponding 11-Z derivatives. In fact, 9-Z-alpha-retinal would be an interesting candidate for retinal supplementation studies. Our data provide direct support for the concept that the 9-methyl group is an important determinant in ligand anchoring and activation of the protein and in general agree with a three-point interaction model involving the ring, 9-methyl group, and aldehyde function.     

Agonists and partial agonists of rhodopsin: retinal polyene methylation affects receptor activation

Using Fourier transform infrared (FTIR) difference spectroscopy, we have studied the impact of sites and extent of methylation of the retinal polyene with respect to position and thermodynamic parameters of the conformational equilibrium between the Meta I and Meta II photoproducts of rhodopsin. Deletion of methyl groups to form 9-demethyl and 13-demethyl analogues, as well as addition of a methyl group at C10 or C12, shifted the Meta I/Meta II equilibrium toward Meta I, such that the retinal analogues behaved like partial agonists. This equilibrium shift resulted from an apparent reduction of the entropy gain of the transition of up to 65%, which was only partially offset by a concomitant reduction of the enthalpy increase. The analogues produced Meta II photoproducts with relatively small alterations, while their Meta I states were significantly altered, which accounted for the aberrant transitions to Meta II. Addition of a methyl group at C14 influenced the thermodynamic parameters but had little impact on the position of the Meta I/Meta II equilibrium. Neutralization of the residue 134 in the E134Q opsin mutant increased the Meta II content of the 13-demethyl analogue, but not of the 9-demethyl analogue, indicating a severe impairment of the allosteric coupling between the conserved cytoplasmic ERY motif involved in proton uptake and the Schiff base/Glu 113 microdomain in the 9-demethyl analogue. The 9-methyl group appears therefore essential for the correct positioning of retinal to link protonation of the cytoplasmic motif with protonation of Glu 113 during receptor activation.     

Depletion of taurine in the adult rat retina

Taurine is the major free amino acid of the vertebrate retina. Treatment of rats with guanidinoethyl sulfonate (GES), a taurine analogue which competes with taurine for transport sites, leads to depletion of 60% of retinal taurine with little effect on other free amino acids. Supplementation of the diet with 0.3% taurine gives partial protection against depletion, confirming that taurine-GES competition underlies part of the effects. The magnitude of the depletion suggests the importance of taurine transport across the blood-retinal barrier for the maintenance of retinal taurine levels.     

Turning cones off: the role of the 9-methyl group of retinal in red cones

Our ability to see in bright light depends critically on the rapid rate at which cone photoreceptors detect and adapt to changes in illumination. This is achieved, in part, by their rapid response termination. In this study, we investigate the hypothesis that this rapid termination of the response in red cones is dependent on interactions between the 9-methyl group of retinal and red cone opsin, which are required for timely metarhodopsin (Meta) II decay. We used single-cell electrical recordings of flash responses to assess the kinetics of response termination and to calculate guanylyl cyclase (GC) rates in salamander red cones containing native visual pigment as well as visual pigment regenerated with 11-cis 9-demethyl retinal, an analogue of retinal in which the 9-methyl group is missing. After exposure to bright light that photoactivated more than approximately 0.2% of the pigment, red cones containing the analogue pigment had a slower recovery of both flash response amplitudes and GC rates (up to 10 times slower at high bleaches) than red cones containing 11-cis retinal. This finding is consistent with previously published biochemical data demonstrating that red cone opsin regenerated in vitro with 11-cis 9-demethyl retinal exhibited prolonged activation as a result of slowed Meta II decay. Our results suggest that two different mechanisms regulate the recovery of responsiveness in red cones after exposure to light. We propose a model in which the response recovery in red cones can be regulated (particularly at high light intensities) by the Meta II decay rate if that rate has been inhibited. In red cones, the interaction of the 9-methyl group of retinal with opsin promotes efficient Meta II decay and, thus, the rapid rate of recovery.     

Effects of Taurine on Calcium Ion Uptake and Protein Phosphorylation in Rat Retinal Membrane Preparations

The effects of taurine on ATP-dependent calcium ion uptake and protein phosphorylation of rat retinal membrane preparations were investigated. Taurine (20 mM) stimulates ATP-dependent calcium ion uptake by twofold in crude retinal homogenates. In contrast, it inhibits the phosphorylation of specific membrane proteins as shown by acrylamide gel electrophoresis and autoradiography. The close structural analogue of taurine, 2-aminoethylhydrogen sulfate, demonstrates similar effects in both systems, i.e., stimulation of ATP-dependent calcium ion uptake and inhibition of protein phosphorylation, whereas isethionic acid and guanidinoethanesulfonate have no effect on either system. A P1 subcellular fraction of the retinal membrane preparation that contains photoreceptor cell synaptosomes has a higher specific activity for the uptake of calcium ions. Phosphorylation of specific proteins in the P1 fraction is also inhibited by the addition of 20 mM taurine. Taurine has no effect on retinal ATPase activities or on phosphatase activity, thus suggesting that it directly affects a kinase system.     

Color regulation in the archaebacterial phototaxis receptor phoborhodopsin (sensory rhodopsin II)

Phoborhodopsin, a repellent phototaxis receptor in Halobacterium halobium, exhibits vibrational fine structure, a feature that has not been identified for any other rhodopsin pigment at physiological temperatures. This conclusion follows form analysis of the absorption properties of the pigment in H. halobium membranes containing native retinal and an array of retinal analogues. The absorption spectrum of the native pigment has a maximum at 487 nm with a pronounced shoulder at 460 nm; however, the bandwidth is that expected for a single retinylidene species. Gaussian band-shape simulation with a spacing corresponding to the vibrational frequencies of polyene stretching modes reproduces the structured absorption spectra of native pigment as well as of analogue phoborhodopsin. Absorption shifts produced by a series of dihydroretinal and other retinal analogues strongly indicate that the dominant factor regulating the color of the pigment is planarization of the retinal ring with respect to the polyene chain.     

The spectral properties and photosensitivities of analogue photopigments regenerated with 10- and 14-substituted retinal analogues

Analogues of 11-cis- and 9-cis-retinal with substitutions at positions 10 and 14 were used to regenerate analogue photopigments with two opsins: that of the transmuted (cone-like) 521-pigment of Gekko gekko and that of the rhodopsin of Porichthys notatus. The spectral absorbances and photosensitivities of the regenerated photopigments were determined and compared, first, between the two systems of analogue photopigments, and second, in the responses to the two opsins. Unlike the 10-fluoropigments, the comparable 14-compounds were significantly red-shifted by 19-30 nm and their sensitivity to light was similar to that of the parent 11-cis- and 9-cis-pigments. These were the results for both analogue pigments. In contrast, the 10-pigments were spectrally located close to the wavelengths of the parent compounds and the photosensitivity was significantly reduced, especially in the case of the 9-cis-analogues. Evidence was obtained for a steric hindrance effect at position 14, for no regeneration was obtained when methyl or ethyl groups were at this carbon. In the 10-substituted retinals, steric hindrance was noted only for the gecko; only the fluorosubstituted, but not the chloro-, the methyl- or the ethyl-substituted, retinals reacted. With the fish opsin, pigments were regenerated with all but the ethyl-substituted retinal. The gecko opsin appears to have a more restricted binding site. Another feature of the gecko was related to the chloride bathochromic and hyperchromic effects, in which the 521-pigment prepared in a chloride-deficient state has a blue-shifted spectrum compared with the spectrum obtained after the addition of chloride, and its extinction is raised by the addition of chloride to give a mean ratio of 1.23 for the two extinctions, one with, the other without, added chloride. The 11-cis-10-F-analogue pigment gave both chloride effects and the hyperchromic ratio was the same as that recorded for the native visual pigment. In contrast, the pigment formed with 11-cis-14-F-retinal gave a hyperchromic ratio significantly greater than 1.23. A similar contrast in the responses to chloride was obtained with the analogue photopigments regenerated with the 9-cis-10-F- and 9-cis-14-F-chromophores. This difference between the two systems is interpreted as the result of a specific configurational feature of the gecko opsin when in the chloride-deficient state that is relevant to the binding of the retinal analogue.(ABSTRACT TRUNCATED AT 400 WORDS)     

The 9-methyl group of retinal is essential for rapid Meta II decay and phototransduction quenching in red cones

Cone photoreceptors of the vertebrate retina terminate their response to light much faster than rod photoreceptors. However, the molecular mechanisms underlying this rapid response termination in cones are poorly understood. The experiments presented here tested two related hypotheses: first, that the rapid decay rate of metarhodopsin (Meta) II in red-sensitive cones depends on interactions between the 9-methyl group of retinal and the opsin part of the pigment molecule, and second, that rapid Meta II decay is critical for rapid recovery from saturation of red-sensitive cones after exposure to bright light. Microspectrophotometric measurements of pigment photolysis, microfluorometric measurements of retinol production, and single-cell electrophysiological recordings of flash responses of salamander cones were performed to test these hypotheses. In all cases, cones were bleached and their visual pigment was regenerated with either 11-cis retinal or with 11-cis 9-demethyl retinal, an analogue of retinal lacking the 9-methyl group. Meta II decay was four to five times slower and subsequent retinol production was three to four times slower in red-sensitive cones lacking the 9-methyl group of retinal. This was accompanied by a significant slowing of the recovery from saturation in cones lacking the 9-methyl group after exposure to bright (>0.1% visual pigment photoactivated) but not dim light. A mathematical model of the turn-off process of phototransduction revealed that the slower recovery of photoresponse can be explained by slower Meta decay of 9-demethyl visual pigment. These results demonstrate that the 9-methyl group of retinal is required for steric chromophore–opsin interactions that favor both the rapid decay of Meta II and the rapid response recovery after exposure to bright light in red-sensitive cones.     

Low aqueous solubility of 11-cis-retinal limits the rate of pigment formation and dark adaptation in salamander rods

We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.     

Lack of interaction of rhodopsin chromophore with membrane lipids. An electron-electron double resonance study using 14N:15N pairs.

Electron-electron double resonance (ELDOR) has been applied to the study of specific interactions of 15N-spin-labeled stearic acid with the retinal chromophore of a rhodopsin analogue containing a 14N spin-labeled retinal. Both the 5 and 16 spin-labeled stearic acids were incorporated into the lipid bilayer of rod outer segment membranes containing the spin-labeled pigment. No interaction between the 15N and 14N spin-labels was observed in rhodopsin or the metarhodopsin II state with either of these labeled stearic acids. Therefore in this system the ring portion of the chromophore must be highly sequestered from the phospholipid bilayer in both the rhodopsin and metarhodopsin II forms.