preparation-living-isolated-vertebrate-photoreceptor-cells-for

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

Calcium (Ca(2+)) modulates several of the enzymatic pathways that mediate phototransduction in the outer segments of vertebrate rod photoreceptors. Ca(2+) enters the rod outer segment through cationic channels kept open by cyclic GMP (cGMP) and is pumped out by a Na(+)/Ca(2+),K(+) exchanger. Light initiates a biochemical cascade, which leads to closure of the cGMP-gated channels, and a concomitant decline in the concentration of Ca(2+). This decline mediates the recovery from stimulation by light and underlies the adaptation of the cell to background light. The speed with which the decline in the Ca(2+) concentration propagates through the rod outer segment depends on the Ca(2+) diffusion coefficient. We have used the fluorescent Ca(2+) indicator fluo-3 and confocal microscopy to measure the profile of the Ca(2+) concentration after stimulation of the rod photoreceptor by light. From these measurements, we have obtained a value of 15 +/- 1 microm(2)s(-1) for the radial Ca(2+) diffusion coefficient. This value is consistent with the effect of a low-affinity, immobile buffer reported to be present in the rod outer segment (L.Lagnado, L. Cervetto, and P.A. McNaughton, 1992, J. Physiol. 455:111-142) and with a buffering capacity of approximately 20 for rods in darkness(S. Nikonov, N. Engheta, and E.N. Pugh, Jr., 1998, J. Gen. Physiol. 111:7-37). This value suggests that diffusion provides a significant delay for the radial propagation of the decline in the concentration of Ca(2+). Also, because of baffling by the disks, the longitudinal Ca(2+) diffusion coefficient will be in the order of 2 microm(2)s(-1), which is much smaller than the longitudinal cGMP diffusion coefficient (30-60 microm(2)s(-1); ). Therefore, the longitudinal decline of Ca(2+) lags behind the longitudinal spread of excitation by cGMP.

Calcium diffusion coefficient in rod photoreceptor outer segments.

Calcium Diffusionskoeffizienten im Stab Photorezeptor &#xE4;u&#xDF;eren Segmente.

Eukaryotic cells use membrane organelles, like the endoplasmic reticulum or the Golgi, to carry out different functions. Vertebrate rod photoreceptors use hundreds of membrane sacs (the disks) for the detection of light. We have used fluorescent tracers and single cell imaging to study the properties of rod photoreceptor disks. Labeling of intact rod photoreceptors with membrane markers and polar tracers revealed communication between intradiskal and extracellular space. Internalized tracers moved along the length of the rod outer segment, indicating communication between the disks as well. This communication involved the exchange of both membrane and aqueous phase and had a time constant in the order of minutes. The communication pathway uses approximately 2% of the available membrane disk area and does not allow the passage of molecules larger than 10 kDa. It was possible to load the intradiskal space with fluorescent Ca(2+) and pH dyes, which reported an intradiskal Ca(2+) concentration in the order of 1 microM and an acidic pH 6.5, both of them significantly different than intracellular and extracellular Ca(2+) concentrations and pH. The results suggest that the rod photoreceptor disks are not discrete, passive sacs but rather comprise an active cellular organelle. The communication between disks may be important for membrane remodeling as well as for providing access to the intradiskal space of the whole outer segment.

Dynamic behavior of rod photoreceptor disks.

Dynamisches Verhalten der Stange Photorezeptor Festplatten.

Visual phototransduction, the conversion of incoming light to an electrical signal, takes place in the outer segments of the rod and cone photoreceptor cells. Light reduces the concentration of cGMP, which, in darkness, keeps open cationic channels present in the plasma membrane of the outer segment. Ca2+ plays an important role in phototransduction by modulating the cGMP-gated channels as well as cGMP synthesis and breakdown. Ca2+ is involved in a negative feedback that is essential for photoreceptor adaptation to background illumination. The effects of Ca2+ on the different components of rod phototransduction have been characterized and can quantitatively account for the steady state responses of the rod cell to background illumination. The propagation of the Ca2+ feedback signal from the periphery toward the center of the outer segment depends on the Ca2+ diffusion coefficient, which has a value of 15 +/- 1 microm2 s(-1). This value shows that diffusion of Ca2+ in the radial direction is quite slow providing a significant barrier in the propagation of the feedback signal. Also, because the diffusion coefficient of Ca2+ is much smaller than that of cGMP, the decline of Ca2+ in the longitudinal direction lags behind the propagation of excitation by the decline of cGMP.

Calcium and phototransduction.

Calcium und Phototransduktion.

Aldehyde dehydrogenase 3A1 (ALDH3A1) is one of the most abundant proteins found in corneal epithelial cells of mammalian species, with several postulated protective roles that include detoxification of peroxidic aldehydes, scavenging of free radicals, and direct absorption of ultraviolet (UV) radiation. In the present study, the protective role of ALDH3A1 against UV- and 4-hydroxy-2-nonenal- (4-HNE-) induced oxidative damage was studied. For this purpose, human ALDH3A1 was stably transfected in a human corneal epithelial cell line (HCE) lacking endogenous enzyme. Cells transfected with ALDH3A1 were more resistant to UV- and 4-HNE-induced cytotoxicity than mock-transfected cells. DNA fragmentation assays revealed that both treatments induced apoptosis in mock-transfected cells, but not in ALDH3A1-expressing cells. Apoptosis appeared to occur via caspase-3 activation and subsequent PARP cleavage. The Michaelis-Menten constant (K(m)) for 4-HNE was 54 microM in ALDH3A1-transfected cells; the addition of 100 microM 4-HNE increased NAD(P)H levels by 50% above that in mock-transfected cells. We also found that ALDH3A1 expression prevented 4-HNE-induced protein adduct formation. Taken together, these data suggest that ALDH3A1 is a regulatory element of the cellular defense system that protects corneal epithelium against UV- and 4-HNE-induced oxidative damage.

Aldh3a1 protects human corneal epithelial cells from ultraviolet- and 4-hydroxy-2-nonenal-induced oxidative damage.

ALDH3A1 Schutz der menschlichen Hornhaut-Epithelzellen von Ultraviolett-und 4-Hydroxy-2-Nonenal-induzierten oxidativen Sch&#xE4;den.

Magnesium ions (Mg2+) play an important role in biochemical functions. In vertebrate photoreceptor outer segments, numerous reactions utilize MgGTP and MgATP, and Mg2+ also regulates several of the phototransduction enzymes. Although Mg2+ can pass through light-sensitive channels under certain conditions, no clear extrusion mechanism has been identified and removing extracellular Mg2+ has no significant effect on the light sensitivity or the kinetics of the photoresponse. We have used the fluorescent Mg2+ dye Furaptra to directly measure and monitor the free Mg2+ concentration in photoreceptor outer segments and examine whether the free Mg2+ concentration changes under physiological conditions. Resting free Mg2+ concentrations in bleached salamander rod and cone photoreceptor cell outer segments were 0.86 +/- 0.06 and 0.81 +/- 0.09 mM, respectively. The outer segment free Mg2+ concentration was not significantly affected by changes in extracellular pH, Ca2+ and Na+, excluding a significant role for the respective exchangers in the regulation of Mg2+ homeostasis. The resting free Mg2+ concentration was also not significantly affected by exposure to 0 Mg2+, suggesting the lack of significant basal Mg2+ flux. Opening the cGMP-gated channels led to a significant increase in the Mg2+ concentration in the absence of Na+ and Ca2+, but not in their presence, indicating that depolarization can cause a significant Mg2+ influx only in the absence of other permeant ions, but not under physiological conditions. Finally, light stimulation did not change the Mg2+ concentration in the outer segments of dark-adapted photoreceptors. The results suggest that there are no influx and efflux pathways that can significantly affect the Mg2+ concentration in the outer segment under physiological conditions. Therefore, it is unlikely that Mg2+ plays a significant role in the dynamic modulation of phototransduction.

Free magnesium concentration in salamander photoreceptor outer segments.

Kostenloser Magnesium-Konzentration im Salamander Photorezeptor &#xE4;u&#xDF;eren Segmente.

Regulation of the visual cycle: retinol dehydrogenase and retinol fluorescence measurements in vertebrate retina.

Verordnung des visuellen Zyklus: Retinol-Dehydrogenase und Retinol Fluoreszenzmessungen in der Wirbeltier-Netzhaut.

The visual cycle comprises a sequence of reactions that regenerate the visual pigment in photoreceptors during dark adaptation, starting with the reduction of all-trans retinal to all-trans retinol and its clearance from photoreceptors. We have followed the reduction of retinal and clearance of retinol within bleached outer segments of red rods isolated from salamander retina by measuring its intrinsic fluorescence. Following exposure to a bright light (bleach), increasing fluorescence intensity was observed to propagate along the outer segments in a direction from the proximal region adjacent to the inner segment toward the distal tip. Peak retinol fluorescence was achieved after approximately 30 min, after which it declined very slowly. Clearance of retinol fluorescence is considerably accelerated by the presence of the exogenous lipophilic substances IRBP (interphotoreceptor retinoid binding protein) and serum albumin. We have used simultaneous fluorometric and electrophysiological measurements to compare the rate of reduction of all-trans retinal to all-trans retinol to the rate of recovery of flash response amplitude in these cells in the presence and absence of IRBP. We find that flash response recovery in rods is modestly accelerated in the presence of extracellular IRBP. These results suggest such substances may participate in the clearance of retinoids from rod photoreceptors, and that this clearance, at least in rods, may facilitate dark adaptation by accelerating the clearance of photoproducts of bleaching.

Physiological and microfluorometric studies of reduction and clearance of retinal in bleached rod photoreceptors.

Physiologische und mikrofluorometrischen Studien der Reduktion und Beseitigung von Retinal in gebleichten St&#xE4;bchen-Photorezeptoren.

The first step in the Visual Cycle, the series of reactions that regenerate the vertebrate visual pigment rhodopsin, is the reduction of all-trans retinal to all-trans retinol, a reaction that requires NADPH. We have used the fluorescence of all-trans retinol to study this reduction in living rod photoreceptors. After the bleaching of rhodopsin, fluorescence (excitation, 360 nm; emission, 457 or 540 nm) appears in frog and wild-type mouse rod outer segments reaching a maximum in 30-60 min at room temperature. With this excitation and emission, the mitochondrial-rich ellipsoid region of the cells shows strong fluorescence as well. Fluorescence measurements at different emission wavelengths establish that the outer segment and ellipsoid signals originate from all-trans retinol and reduced pyridine nucleotides, respectively. Using outer segment fluorescence as a measure of all-trans retinol formation, we find that in frog rod photoreceptors the NADPH necessary for the reduction of all-trans retinal can be supplied by both cytoplasmic and mitochondrial metabolic pathways. Inhibition of the reduction reaction, either by retinoic acid or through suppression of metabolic activity, reduced the formation of retinol. Finally, there are no significant fluorescence changes after bleaching in the rod outer segments of Rpe65(-/-) mice, which lack 11-cis retinal.

Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors.

Reduzierung der all-trans-Retinal zu all-trans-Retinol in den &#xE4;u&#xDF;eren Segmenten der Frosch und Maus-St&#xE4;bchen-Photorezeptoren.

Vertebrate rod photoreceptors are the ultimate light sensors, as they can detect a single photon. In darkness, rods maintain a high concentration of the intracellular messenger cyclic guanosine monophosphate (cGMP), which binds to and keeps open cationic channels on the plasma membrane of the outer segment. Absorption of a photon by the visual pigment of the rod, rhodopsin, initiates a biochemical amplification cascade that leads to a reduction in the concentration of cGMP and closure of the channels, thereby converting the incoming light to an electrical signal. Because the absorption of a photon and the ensuing reactions are localized events, the magnitude of the response of the rod to a single photon depends on the spread of the decrease in the cGMP concentration along the length of the outer segment. The longitudinal diffusion of cGMP depends on the structural parameters of the rod outer segment, specifically the area and the volume available for diffusion. To characterize the effect of rod outer segment cytoarchitecture on diffusion, we have used fluorescence recovery after photobleaching (FRAP) and examined the mobility of a fluorescent polar tracer, calcein, in the rod outer segments from three species with different outer segment structures: frog (Rana pipiens), mouse (Mus musculus domesticus) and gecko (Gekko gekko). We found that the diffusion coefficient is similar for all three species, in the order of 8-17 microm(2) s(-1), in broad agreement with the predictions by Holcman and Korenbrot (Biophys. J. 2004:86;2566-2582) based on the known cytoarchitecture of rod outer segments. Consequently, the results also support their prediction that the longitudinal spread of light excitation in rods is similar across species.

Longitudinal diffusion of a polar tracer in the outer segments of rod photoreceptors from different species.

Vertebrate Stab Photorezeptoren sind die ultimativen Lichtsensoren, wie sie ein einzelnes Photon erkennen k&ouml;nnen. In der Dunkelheit pflegen die St&auml;be eine hohe Konzentration an die intrazellul&auml;re Messenger cyclisches Guanosinmonophosphat (cGMP), die bindet an und h&auml;lt offene kationische Kan&auml;le auf der Plasmamembran im &auml;u&szlig;eren Segment. Absorption eines Photons durch das Sehpigment des Stabes, Rhodopsin, initiiert eine biochemische Verst&auml;rkung-Kaskade, das f&uuml;hrt zu einer Verringerung der Konzentration von cGMP und Schlie&szlig;ung der Kan&auml;le, damit das einfallende Licht in elektrische Signale umwandeln. Da die Absorption eines Photons und die darauf folgenden Reaktionen sind lokalisierte Ereignisse, h&auml;ngt das Ausma&szlig; der Reaktion des Stabes auf ein einzelnes Photon die Ausbreitung der R&uuml;ckgang der cGMP-Konzentration entlang der L&auml;nge des &auml;u&szlig;eren Segment. Die longitudinale Diffusion von cGMP, h&auml;ngt von der Strukturparameter von &auml;u&szlig;eren Segment der Stab, speziell die Fl&auml;chen- und die Verbreitung zur Verf&uuml;gung. Um die Wirkung der Stab &auml;u&szlig;ere Segment Cytoarchitecture Diffusion zu charakterisieren, haben wir Fluoreszenz-Wiederherstellung nach Photobleaching (FRAP) verwendet und die Mobilit&auml;t von einem fluoreszierenden polar Tracer, Calcein, in die &auml;u&szlig;eren Stab-Segmente aus drei Arten mit verschiedenen &auml;u&szlig;eren Segment Strukturen untersucht: Frosch (Rana Pipiens), Maus (Mus Musculus Domesticus) und Gecko (Gekko Gekko). Wir festgestellt, dass der Diffusionskoeffizient f&uuml;r alle drei Arten, in der Gr&ouml;&szlig;enordnung von 8-17-microm(2)-s(-1), &auml;hnlich in breite &Uuml;bereinstimmung mit den Vorhersagen von Holcman und Korenbrot (Biophys. J. 2004:86; 2566-2582) basierend auf den bekannten Cytoarchitecture Stab &auml;u&szlig;eren Segmente. Daher unterst&uuml;tzen die Ergebnisse auch ihre Vorhersage, dass die L&auml;nge Ausbreitung von Licht Erregung in Stangen Arten &auml;hnlich ist.

Longitudinale Diffusion ein polar Tracer in die &auml;u&szlig;eren Segmente der Stab Photorezeptoren von verschiedenen Arten.

The visual pigment protein of vertebrate rod photoreceptors, rhodopsin, contains an 11-cis retinyl moiety that is isomerized to all-trans upon light absorption. Subsequently, all-trans retinal is released from the protein and reduced to all-trans retinol, the first step in the recycling of rhodopsin's chromophore group through the series of reactions that constitute the visual cycle. The concentration of all-trans retinol in photoreceptor outer segments can be monitored from its fluorescence. We have used two-photon excitation (720 nm) of retinol fluorescence and fluorescence recovery after photobleaching to characterize the mobility of all-trans retinol in frog photoreceptor outer segments. Retinol produced after rhodopsin bleaching moved laterally in the disk membrane bilayer with an apparent diffusion coefficient of 2.5 +/- 0.3 micro m(2) s(-1). The diffusion coefficient of exogenously added retinol was 3.2 +/- 0.5 micro m(2) s(-1). These diffusion coefficients are in close agreement with those reported for lipids, suggesting that retinol is not tightly bound to protein sites that would be diffusing much more slowly in the plane of the membrane. In agreement with this interpretation, a fluorescent-labeled C-16 fatty acid diffused laterally with a similar diffusion coefficient, 2.2 +/- 0.2 micro m(2) s(-1). Retinol also moved along the length of the rod outer segment, with an apparent diffusion coefficient of 0.07 +/- 0.01 micro m(2) s(-1), again suggesting that it is not tightly bound to proteins that would confine it to the disks. The axial diffusion coefficient of exogenously added retinol was 0.05 +/- 0.01 micro m(2) s(-1). In agreement with passive diffusion, the rate of axial movement was inversely proportional to the square of the length of the rod outer segment. Diffusion of retinol on the plasma membrane of the outer segment can readily account for the measured value of the axial diffusion coefficient, as the plasma membrane comprises approximately 1% of the total outer-segment membrane. The values of both the lateral and axial diffusion coefficients are consistent with most of the all-trans retinol in the outer segments moving unrestricted and not being bound to carrier proteins. Therefore, and in contrast to other steps of the visual cycle, there does not appear to be any specialized processing for all-trans retinol within the rod outer segment.

All-trans retinol in rod photoreceptor outer segments moves unrestrictedly by passive diffusion.

Das Sehpigment Protein von vertebrate Stab Lichtrezeptoren, Rhodopsin, enth&auml;lt ein 11-Cis-Retinilpalmitat-Gruppe, die zu All-Trans nach Lichtabsorption isomerized ist. All-Trans-Retinal ist anschlie&szlig;end freigegeben vom Protein und reduzierten zu All-Trans Retinol, der erste Schritt im recycling von Rhodopsin der Chromophor-Gruppe durch die Serie von Reaktionen, die den visuellen Zyklus bilden. Die Konzentration von All-Trans Retinol in Photorezeptoren &auml;u&szlig;eren Segmente kann von ihrer Fluoreszenz &uuml;berwacht werden. Wir haben zwei-Photonen-Anregung verwendet (720 nm) Retinol Fluoreszenz und Fluoreszenz Recovery after Photobleaching, die Mobilit&auml;t von All-Trans Retinol Frosch Photorezeptor &auml;u&szlig;eren Segmente zu charakterisieren. Retinol produziert, nachdem die Festplatte Membran Bilayer mit eine scheinbare Diffusionskoeffizient 2,5 +/-0,3 Mikro m s(-1) Rhodopsin bleichen seitlich verlegt werden. Der Diffusionskoeffizient von exogen hinzugef&uuml;gt Retinol war 3.2 +/-0,5 m Mikro-s(-1). Diese Diffusionskoeffizienten sind in enger Abstimmung mit den berichteten f&uuml;r Lipide, was darauf hindeutet, dass dieses Retinol nicht eng an Protein-Standorte gebunden ist, die sehr viel langsamer in der Ebene der Membran zu einer Diffusion werden w&uuml;rde. Im Einvernehmen mit dieser Interpretation verbreitet eine Fluoreszenz-markierten C-16-Fetts&auml;ure seitlich mit einer &auml;hnlichen Diffusionskoeffizient, 2.2 +/-0,2 Mikro m s(-1). Retinol, zog auch entlang der L&auml;nge des Segments &auml;u&szlig;eren Stab mit einer scheinbaren Diffusionskoeffizienten von 0,07 +/-0.01 Mikro m s(-1), wieder darauf hindeutet, dass es nicht eng an Proteine gebunden ist, die sie sich auf den Datentr&auml;gern beschr&auml;nken w&uuml;rde. Die axiale Diffusionskoeffizient von exogen hinzugef&uuml;gt Retinol war 0,05 +/-0.01 Mikro m s(-1). Im Einvernehmen mit passive Diffusion war die Rate der axiale Bewegung umgekehrt proportional zum Quadrat der L&auml;nge des Segments &auml;u&szlig;eren Stab. Verbreitung von Retinol auf der Plasmamembran im &auml;u&szlig;eren Segment kann leicht f&uuml;r den Messwert f&uuml;r die axiale Diffusionskoeffizient ber&uuml;cksichtigen, wie die Plasmamembran etwa 1 % der gesamten &auml;u&szlig;eren-Segment-Membran umfasst. Die Werte f&uuml;r die laterale und axiale Diffusionskoeffizienten entsprechen die meisten All-Trans-Retinol in die &auml;u&szlig;eren Segmente verschieben eine uneingeschr&auml;nkte und nicht an Carrier-Proteine gebunden wird. Daher, und im Gegensatz zu anderen Schritte des visuellen Zyklus, erscheint es keine spezielle Verarbeitung f&uuml;r All-Trans Retinol im &auml;u&szlig;eren Segment Stab sein.

All-Trans Retinol im Stab Photorezeptor &auml;u&szlig;eren Segmente bewegt sich uneingeschr&auml;nkt durch passive Diffusion.

To test whether the formation of all-trans retinol limits the regeneration of the visual pigment. all-trans retinol is formed after visual pigment bleaching through the reduction of all-trans retinal in a reaction involving NADPH. This reduction begins the recycling of the chromophore for the regeneration of the visual pigment.

Formation of all-trans retinol after visual pigment bleaching in mouse photoreceptors.

Testen Sie, ob die Bildung von All-Trans-Retinol die Regeneration das Sehpigment begrenzt. All-Trans-Retinol wird nach dem Sehpigment bleichen durch die Verringerung von All-Trans-Retinal in einer Reaktion mit NADPH gebildet. Diese Reduktion beginnt das recycling von den Chromophor zur Regeneration von dem Sehpigment.

Bildung von All-Trans-Retinol nach Sehpigment bleichen in Maus Photorezeptoren.

To determine whether a nonprotein lipophilic carrier, 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), can remove all-trans retinol from rod photoreceptor outer segments. All-trans retinol is generated in rod outer segments after light exposure. It is highly insoluble, and its efficient transport across extra- and intracellular aqueous space requires specialized carriers.

2-Hydroxypropyl-beta-cyclodextrin removes all-trans retinol from frog rod photoreceptors in a concentration-dependent manner.

Um festzustellen, ob ein Nonprotein lipophile Tr&auml;ger, k&ouml;nnen 2-Hydroxypropyl-Beta-Cyclodextrin (HP-Beta-CD), All-Trans-Retinol aus entfernen Stab Photorezeptor &auml;u&szlig;eren Segmente. All-Trans-Retinol wird im Stab &auml;u&szlig;eren Segmente nach Lichtexposition generiert. Es ist sehr schwer wasserl&ouml;slich und der effiziente Transport &uuml;ber extra- und intrazellul&auml;re w&auml;ssrigen Raum erfordert spezielle Tr&auml;ger.

2-Hydroxypropyl-Beta-Cyclodextrin entfernt Frosch Stab Photorezeptoren Konzentration-abh&auml;ngigen Weise All-Trans-Retinol.

All-trans retinol is formed in the outer segments of vertebrate rod photoreceptors from the reduction of the all-trans retinal released by photoactivated rhodopsin. The reduction requires NADPH and is therefore dependent on metabolic input. In metabolically intact photoreceptors, a large increase in rod outer segment fluorescence, attributed to the fluorescence of all-trans retinol, follows rhodopsin photoactivation. The fluorescence increase is biphasic, including a rapid and a slow component. In metabolically compromised cells, there is a much smaller fluorescence increase following rhodopsin photoactivation, but it too contains a rapid component. We have measured the fluorescence signal in single living frog and mouse rod photoreceptors, and have characterized its dependence on the wavelengths of light selected for excitation and for collecting emission. We find that in metabolically intact cells, the excitation and emission properties of both the rapid and slow components of the fluorescence signal are in close agreement with those of all-trans retinol fluorescence. In metabolically compromised cells, however, the signal can only partially be due to all-trans retinol, and most of it is consistent with all-trans retinal. The results suggest that in the outer segments of living rod photoreceptors there is rapid release of all-trans retinal, which in metabolically intact cells is accompanied by rapid conversion to all-trans retinol.

Rapid formation of all-trans retinol after bleaching in frog and mouse rod photoreceptor outer segments.

All-Trans-Retinol ist die &auml;u&szlig;eren Segmente des vertebrate Stab Photorezeptoren aus der Reduzierung der All-Trans-Retinal, ver&ouml;ffentlicht von Photoactivated Rhodopsin gebildet. Die Reduktion NADPH ben&ouml;tigt und ist daher abh&auml;ngig von metabolischen Eingang. In metabolisch intakten Photorezeptoren eine gro&szlig;e Zunahme der Stab &auml;u&szlig;ere Segment Fluoreszenz, zur&uuml;ckzuf&uuml;hren auf die Fluoreszenz von All-Trans-Retinol, Rhodopsin Photoaktivierung folgt. Die Fluoreszenz-Erh&ouml;hung wird biphasische, z. B. eine schnelle und eine langsame Komponente. In metabolisch befallenen Zellen, gibt es eine viel kleinere Fluoreszenz-Zuwachs nach Rhodopsin Photoaktivierung, aber es enth&auml;lt auch eine rasche Komponente. Wir haben gemessen, das Fluoreszenz-Signal im einzigen lebenden Frosch und Maus Stab Photorezeptoren und seine Abh&auml;ngigkeit von den Wellenl&auml;ngen des Lichts, die ausgew&auml;hlte f&uuml;r Erregung und zum Sammeln von Emission gepr&auml;gt haben. Wir finden, dass in metabolisch intakten Zellen, die Anregungs- und Eigenschaften der schnellen und langsamen Komponenten der das Fluoreszenz-Signal in enger Abstimmung mit denen des All-Trans Retinol Fluoreszenz. In metabolisch befallenen Zellen jedoch das Signal kann nur teilweise an den All-Trans-Retinol liegen, und das meiste davon steht im Einklang mit All-Trans-Retinal. Die Ergebnisse legen nahe, dass in die &auml;u&szlig;eren Segmente des Lebens Stab Photorezeptoren gibt es schnelle Freisetzung von All-Trans-Retinal, die in metabolisch intakte Zellen durch rasche Umstellung auf All-Trans-Retinol begleitet wird.

Schnelle Bildung von All-Trans-Retinol nach Frosch Maus Stab Photorezeptor &auml;u&szlig;eren Segmente und Bleichen.

The reactive aldehyde all-trans retinal is released in rod photoreceptor outer segments by photoactivated rhodopsin and is eliminated through reduction to all-trans retinol. This study was undertaken to determine whether all-trans retinol formation depends on Abca4, arrestin, rhodopsin kinase, and the palmitylation of rhodopsin, all of which are factors that affect the release and sequestration of all-trans retinal.

Rod outer segment retinol formation is independent of Abca4, arrestin, rhodopsin kinase, and rhodopsin palmitylation.

Die reaktive Aldehyde All-Trans-Retinal wird in Stab Photorezeptor &auml;u&szlig;eren Segmente von Photoactivated Rhodopsin und durch Reduktion auf All-Trans-Retinol beseitigt ist. Diese Studie wurde durchgef&uuml;hrt, um festzustellen, ob All-Trans Retinol Bildung Abca4 h&auml;ngt, arrestin, Rhodopsin-Kinase und die Palmitylation des Rhodopsin, die Faktoren sind, die Ver&ouml;ffentlichung und die Beschlagnahme von All-Trans-Retinal auswirken.

Article	Year
Calcium diffusion coefficient in rod photoreceptor outer segments. Biophysical journal\| PubMed ID: 11806915	2002
Dynamic behavior of rod photoreceptor disks. Biophysical journal\| PubMed ID: 12202366	2002
Calcium and phototransduction. Advances in experimental medicine and biology\| PubMed ID: 12596912	2002
Aldh3a1 protects human corneal epithelial cells from ultraviolet- and 4-hydroxy-2-nonenal-induced oxidative damage. Free radical biology & medicine\| PubMed ID: 12706498	2003
Free magnesium concentration in salamander photoreceptor outer segments. The Journal of physiology\| PubMed ID: 14500766	2003
Regulation of the visual cycle: retinol dehydrogenase and retinol fluorescence measurements in vertebrate retina. Advances in experimental medicine and biology\| PubMed ID: 15180285	2003
Physiological and microfluorometric studies of reduction and clearance of retinal in bleached rod photoreceptors. The Journal of general physiology\| PubMed ID: 15452202	2004
Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors. Biophysical journal\| PubMed ID: 15626704	2005
Longitudinal diffusion of a polar tracer in the outer segments of rod photoreceptors from different species. Photochemistry and photobiology\| PubMed ID: 16906792	2006
All-trans retinol in rod photoreceptor outer segments moves unrestrictedly by passive diffusion. Biophysical journal\| PubMed ID: 17012326	2006
Formation of all-trans retinol after visual pigment bleaching in mouse photoreceptors. Investigative ophthalmology & visual science\| PubMed ID: 19264891	2009
2-Hydroxypropyl-beta-cyclodextrin removes all-trans retinol from frog rod photoreceptors in a concentration-dependent manner. Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics\| PubMed ID: 20565310	2010
Rapid formation of all-trans retinol after bleaching in frog and mouse rod photoreceptor outer segments. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology\| PubMed ID: 20697621	2010
Rod outer segment retinol formation is independent of Abca4, arrestin, rhodopsin kinase, and rhodopsin palmitylation. Investigative ophthalmology & visual science\| PubMed ID: 21398289	2011

Chunhe Chen

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