The First Steps of the Visual Cycle in Human Rod and Cone Photoreceptors.

Purpose: Light detection destroys the visual pigment. Its regeneration, necessary for the recovery of light sensitivity, is accomplished through the visual cycle. Release of all-trans retinal by the light-activated visual pigment and its reduction to all-trans retinol comprise the first steps of the visual cycle. In this study, we determined the kinetics of all-trans retinol formation in human rod and cone photoreceptors. Methods: Single living rod and cone photoreceptors were isolated from the retinas of human cadaver eyes (ages 21 to 90 years). Formation of all-trans retinol was measured by imaging its outer segment fluorescence (excitation, 360 nm; emission, >420 nm). The extent of conversion of released all-trans retinal to all-trans retinol was determined by measuring the fluorescence excited by 340 and 380 nm. Measurements were repeated with photoreceptors isolated from Macaca fascicularis retinas. Experiments were carried out at 37°C. Results: We found that ∼80% to 90% of all-trans retinal released by the light-activated pigment is converted to all-trans retinol, with a rate constant of 0.24 to 0.55 min-1 in human rods and ∼1.8 min-1 in human cones. In M. fascicularis rods and cones, the rate constants were 0.38 ± 0.08 min-1 and 4.0 ± 1.1 min-1, respectively. These kinetics are several times faster than those measured in other vertebrates. Interphotoreceptor retinoid-binding protein facilitated the removal of all-trans retinol from human rods. Conclusions: The first steps of the visual cycle in human photoreceptors are several times faster than in other vertebrates and in line with the rapid recovery of light sensitivity exhibited by the human visual system.

Interphotoreceptor retinoid-binding protein removes all-trans-retinol and retinal from rod outer segments, preventing lipofuscin precursor formation.

Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of retinal and retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-retinal is reduced by the retinol dehydrogenase RDH8 to all-trans-retinol in an NADPH-dependent reaction. However, all-trans-retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-retinol, all-trans-retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 µm-1 IRBP also removed all-trans-retinal, but with much less efficacy, indicating that the reduction of retinal to retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-retinal and retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-retinal.

All-trans retinal levels and formation of lipofuscin precursors after bleaching in rod photoreceptors from wild type and Abca4-/- mice.

The accumulation of lipofuscin in the cells of the retinal pigment epithelium (RPE) is thought to play an important role in the development and progression of degenerative diseases of the retina. The bulk of RPE lipofuscin originates in reactions of the rhodopsin chromophore, retinal, with components of the photoreceptor outer segment. The 11-cis retinal isomer is generated in the RPE and supplied to rod photoreceptor outer segments where it is incorporated as the chromophore of rhodopsin. It is photoisomerized during light detection to all-trans and subsequently released by photoactivated rhodopsin as all-trans retinal, which is removed through reduction to all-trans retinol in a reaction requiring metabolic input in the form of NADPH. Both 11-cis and all-trans retinal can form lipofuscin precursor fluorophores in rod photoreceptor outer segments. Increased accumulation of lipofuscin has been suggested to result from excess formation of lipofuscin precursors due to buildup of all-trans retinal released by light exposure. In connection with this suggestion, the Abca4 transporter protein, an outer segment protein defects in which result in recessive Stargardt disease, has been proposed to promote the removal of all-trans retinal by facilitating its availability for reduction. To examine this possibility, we have measured the outer segment levels of all-trans retinal, all-trans retinol, and of lipofuscin precursors after bleaching by imaging the fluorescence of single rod photoreceptors isolated from wild type and Abca4-/- mice. We found that all-trans retinol and all-trans retinal levels increased after bleaching in both wild type and Abca4-/- rods. At all times after bleaching, there was no significant difference in all-trans retinal levels between the two strains. All-trans retinol levels were not significantly different between the two strains at early times, but were lower in Abca4-/- rods at times longer than 20 min after bleaching. Bleaching in the presence of lower metabolic substrate concentrations resulted in higher all-trans retinal levels and increased formation of lipofuscin precursors in both wild type and Abca4-/- rods. The results show that conditions that result in buildup of all-trans retinal levels result in increased generation of lipofuscin precursors in both wild type and Abca4-/- rods. The results are consistent with the proposal that Abca4 facilitates the reduction of all-trans retinal to retinol; absence of Abca4 however does not appear to be associated with higher all-trans retinal levels compared to wild type.

Rhodopsin kinase and arrestin binding control the decay of photoactivated rhodopsin and dark adaptation of mouse rods.

Photoactivation of vertebrate rhodopsin converts it to the physiologically active Meta II (R*) state, which triggers the rod light response. Meta II is rapidly inactivated by the phosphorylation of C-terminal serine and threonine residues by G-protein receptor kinase (Grk1) and subsequent binding of arrestin 1 (Arr1). Meta II exists in equilibrium with the more stable inactive form of rhodopsin, Meta III. Dark adaptation of rods requires the complete thermal decay of Meta II/Meta III into opsin and all-trans retinal and the subsequent regeneration of rhodopsin with 11-cis retinal chromophore. In this study, we examine the regulation of Meta III decay by Grk1 and Arr1 in intact mouse rods and their effect on rod dark adaptation. We measure the rates of Meta III decay in isolated retinas of wild-type (WT), Grk1-deficient (Grk1(-/-)), Arr1-deficient (Arr1(-/-)), and Arr1-overexpressing (Arr1(ox)) mice. We find that in WT mouse rods, Meta III peaks ∼6 min after rhodopsin activation and decays with a time constant (τ) of 17 min. Meta III decay slows in Arr1(-/-) rods (τ of ∼27 min), whereas it accelerates in Arr1(ox) rods (τ of ∼8 min) and Grk1(-/-) rods (τ of ∼13 min). In all cases, regeneration of rhodopsin with exogenous 11-cis retinal is rate limited by the decay of Meta III. Notably, the kinetics of rod dark adaptation in vivo is also modulated by the levels of Arr1 and Grk1. We conclude that, in addition to their well-established roles in Meta II inactivation, Grk1 and Arr1 can modulate the kinetics of Meta III decay and rod dark adaptation in vivo.

Determination of N-retinylidene-N-retinylethanolamine (A2E) levels in central and peripheral areas of human retinal pigment epithelium.

The bis-retinoid N-retinylidene-N-retinylethanolamine (A2E) is one of the major components of lipofuscin, a fluorescent material that accumulates with age in the lysosomes of the retinal pigment epithelium (RPE) of the human eye. Lipofuscin, as well as A2E, exhibit a range of cytotoxic properties, which are thought to contribute to the pathogenesis of degenerative diseases of the retina such as Age-related Macular Degeneration. Consistent with such a pathogenic role, high levels of lipofuscin fluorescence are found in the central area of the human RPE, and decline toward the periphery. Recent reports have however suggested a surprising incongruence between the distributions of lipofuscin and A2E in the human RPE, with A2E levels being lowest in the central area and increasing toward the periphery. To appraise such a possibility, we have quantified the levels of A2E in the central and peripheral RPE areas of 10 eyes from 6 human donors (ages 75-91 years) with HPLC and UV/VIS spectroscopy. The levels of A2E in the central area were on average 3-6 times lower than in peripheral areas of the same eye. Furthermore, continuous accumulation of selected ions (CASI) imaging mass spectrometry showed the presence of A2E in the central RPE, and at lower intensities than in the periphery. We have therefore corroborated that in human RPE the levels of A2E are lower in the central area compared to the periphery. We conclude that the levels of A2E cannot by themselves provide an explanation for the higher lipofuscin fluorescence found in the central area of the human RPE.

The 11-cis Retinal Origins of Lipofuscin in the Retina.

Lipofuscin is a fluorescent mixture of partially digested proteins and lipids that accumulates with age in the lysosomal compartment of the retinal pigment epithelium (RPE) of the eye. Because it has been found to have significant cytotoxic potential, lipofuscin is thought to play a role in retinal degeneration diseases including age-related macular degeneration and Stargardt disease, a form of juvenile macular degeneration. The only known components of lipofuscin are bis-retinoids, the condensation products of two molecules of retinal. The bulk of lipofuscin is thought to originate in the rod photoreceptor outer segments as a by-product of reactions involving the retinal chromophore of rhodopsin. 11-cis retinal flows from the RPE into the rod outer segments, where it combines with opsin to form rhodopsin; all-trans retinal is released into the rod outer segments by photoactivated rhodopsin following its excitation by light. Both 11-cis and all-trans retinal can generate lipofuscin-like fluorophores and bis-retinoids when added to rod outer segment membranes. The levels of lipofuscin precursor fluorophores present in the outer segments of dark-adapted rods are similar in cyclic-light- and dark-reared mice, as are the levels of accumulated lipofuscin in the RPE. Because the retinol dehydrogenase enzyme present in rod outer segments can reduce all-trans but not 11-cis retinal, lipofuscin precursors are more likely to form from 11-cis than all-trans retinal, even under cyclic light conditions. Thus, 11-cis retinal may be the primary source of lipofuscin in the retina.

Kinetics of rhodopsin's chromophore monitored in a single photoreceptor.

Absorption of light isomerizes the retinyl chromophore of the photoreceptor pigment rhodopsin from 11-cis to all-trans, generating the photoactivated rhodopsin form. The photoisomerization of the chromophore however destroys rhodopsin, and its regeneration requires the removal of the all-trans and the supply of fresh 11-cis chromophore. The all-trans chromophore is removed through a series of steps beginning with its release from photoactivated rhodopsin in the form of all-trans-retinal, leaving behind the apoprotein opsin. All-trans-retinal is then reduced to all-trans-retinol, which is transported out of the photoreceptor. Rhodopsin is regenerated from opsin and fresh 11-cis-retinal arriving to the photoreceptor from the retinal pigment epithelium. Both all-trans and 11-cis-retinal can form precursors of lipofuscin, a pigment that accumulates with age in the lysosomal compartment of the retinal pigment epithelium. All-trans-retinal, all-trans-retinol, and lipofuscin precursors all emit significant and distinct fluorescence signals, allowing their monitoring in single photoreceptor cells with fluorescence imaging. Here we describe the procedures for measuring these fluorophores in single mouse rod photoreceptors.

Mitochondria contribute to NADPH generation in mouse rod photoreceptors.

NADPH is the primary source of reducing equivalents in the cytosol. Its major source is considered to be the pentose phosphate pathway, but cytosolic NADP(+)-dependent dehydrogenases using intermediates of mitochondrial pathways for substrates have been known to contribute. Photoreceptors, a nonproliferating cell type, provide a unique model for measuring the functional utilization of NADPH at the single cell level. In these cells, NADPH availability can be monitored from the reduction of the all-trans-retinal generated by light to all-trans-retinol using single cell fluorescence imaging. We have used mouse rod photoreceptors to investigate the generation of NADPH by different metabolic pathways. In the absence of extracellular metabolic substrates, NADPH generation was severely compromised. Extracellular glutamine supported NADPH generation to levels comparable to those of glucose, but pyruvate and lactate were relatively ineffective. At low extracellular substrate concentrations, partial inhibition of ATP synthesis lowered, whereas suppression of ATP consumption augmented NADPH availability. Blocking pyruvate transport into mitochondria decreased NADPH availability, and addition of glutamine restored it. Our findings demonstrate that in a nonproliferating cell type, mitochondria-linked pathways can generate substantial amounts of NADPH and do so even when the pentose phosphate pathway is operational. Competing demands for ATP and NADPH at low metabolic substrate concentrations indicate a vulnerability to nutrient shortages. By supporting substantial NADPH generation, mitochondria provide alternative metabolic pathways that may support cell function and maintain viability under transient nutrient shortages. Such pathways may play an important role in protecting against retinal degeneration.

Sepsis mortality prediction based on predisposition, infection and response.

OBJECTIVE: To empirically test, based on a large multicenter, multinational database, whether a modified PIRO (predisposition, insult, response, and organ dysfunction) concept could be applied to predict mortality in patients with infection and sepsis. DESIGN: Substudy of a multicenter multinational cohort study (SAPS 3). PATIENTS: A total of 2,628 patients with signs of infection or sepsis who stayed in the ICU for >48 h. Three boxes of variables were defined, according to the PIRO concept. Box 1 (Predisposition) contained information about the patient's condition before ICU admission. Box 2 (Injury) contained information about the infection at ICU admission. Box 3 (Response) was defined as the response to the infection, expressed as a Sequential Organ Failure Assessment score after 48 h. INTERVENTIONS: None. MAIN MEASUREMENTS AND RESULTS: Most of the infections were community acquired (59.6%); 32.5% were hospital acquired. The median age of the patients was 65 (50-75) years, and 41.1% were female. About 22% (n=576) of the patients presented with infection only, 36.3% (n=953) with signs of sepsis, 23.6% (n=619) with severe sepsis, and 18.3% (n=480) with septic shock. Hospital mortality was 40.6% overall, greater in those with septic shock (52.5%) than in those with infection (34.7%). Several factors related to predisposition, infection and response were associated with hospital mortality. CONCLUSION: The proposed three-level system, by using objectively defined criteria for risk of mortality in sepsis, could be used by physicians to stratify patients at ICU admission or shortly thereafter, contributing to a better selection of management according to the risk of death.

High-dose remifentanil does not impair cerebrovascular carbon dioxide reactivity in healthy male volunteers.

BACKGROUND: Cerebrovascular carbon dioxide reactivity during high-dose remifentanil infusion was investigated in volunteers by measurement of regional cerebral blood flow (rCBF) and mean CBF velocity (CBFv). METHODS: Ten healthy male volunteers with a laryngeal mask for artificial ventilation received remifentanil at an infusion rate of 2 and 4 microg x kg-1 x min-1 under normocapnia, hypocapnia, and hypercapnia. Stable xenon-enhanced computed tomography and transcranial Doppler ultrasonography of the left middle cerebral artery were used to assess rCBF and mean CBFv, respectively. If required, blood pressure was maintained within baseline values with intravenous phenylephrine to avoid confounding effects of altered hemodynamics. RESULTS: Hemodynamic parameters were maintained constant over time. Remifentanil infusion at 2 and 4 microg x kg-1 x min-1 significantly decreased rCBF and mean CBFv. Both rCBF and mean CBFv increased as the arterial carbon dioxide tension increased from hypocapnia to hypercapnia, indicating that cerebrovascular reactivity remained intact. The average slopes of rCBF reactivity were 0.56 +/- 0.27 and 0.49 +/- 0.28 ml. 100 g-1 x min-1 x mmHg-1 for 2 and 4 microg x kg-1 x min-1 remifentanil, respectively (relative change in percent/mmHg: 1.9 +/- 0.8 and 1.6 +/- 0.5, respectively). The average slopes for mean CBFv reactivity were 1.61 +/- 0.95 and 1.54 +/- 0.83 cm x s-1 x mmHg-1 for 2 and 4 microg x kg-1 x min-1 remifentanil, respectively (relative change in percent/mmHg: 1.86 +/- 0.59 and 1.79 +/- 0.59, respectively). Preanesthesia and postanesthesia values of rCBF and mean CBFv did not differ. CONCLUSION: High-dose remifentanil decreases rCBF and mean CBFv without impairing cerebrovascular carbon dioxide reactivity. This, together with its known short duration of action, makes remifentanil a useful agent in the intensive care unit when sedation that can be titrated rapidly is required.