Teratogenicity, tissue distribution, and metabolism of the retro-retinoids, 14-hydroxy-4,14-retro-retinol and anhydroretinol, in the C57BL/6J mouse.

The retro-retinoids 14-hydroxy-4,14-retro-retinol (14-HRR) and anhydroretinol (AR) are endogenous metabolites of retinol (Vitamin A). 14-HRR and retinol, but not retinoic acid, promote the proliferation of lymphocytes and fibroblasts when cultured in serum-free medium, whereas AR competitively inhibits these growth-supportive effects. Retinol and all-trans-retinoic acid are potent teratogens. This study shows the teratogenic potencies of 14-HRR and AR compared to retinol at a single gestational time. Also reported is the metabolism of these retinoids in nonpregnant mouse liver, the primary storage tissue of vitamin A, where many retinoids will be present at their highest concentration. Additionally, measurement of these metabolite concentrations was carried out in pregnant mouse plasma and embryos because they are the most relevant to teratology. Single intraperitoneal administration of 60 mg/kg of all-trans-retinol (retinol) to C57BL/6J mice at gestational day 7.5 produced a significant induction of eye and axial skeletal malformations. The equivalent dose of 14-HRR or AR induced a lower frequency of embryolethality and eye and axial skeletal malformations indicating that these retro-retinoids are less potent teratogens than retinol. The distribution of 14-HRR, AR, retinol, and their metabolites was determined in the liver at a single time point after retinoid administration. Two hours after 60 mg/kg of 14-HRR treatment, HRR esters are detected. Two hours after 600 mg/kg of AR treatment, 14-HRR is detected, suggesting that 14-HRR, a reported metabolite of retinol, can be biosynthesized from AR. In both cases, neither retinoic acid nor retro-retinoid acidic metabolites were detected. Two hours after 60 mg/kg of retinol treatment, 14-HRR, 13,14-dihydroxyretinol (DHR), AR, and retinoic acid were detected. A new endogenous retro-retinoid, to which the 4-hydro-5-hydroxy-anhydroretinol structure is proposed, was detected in all liver extracts. Retinoic acid, 14-HRR, and DHR were present in plasma and embryos of retinol-treated pregnant mice. Plasma and embryos of AR-treated pregnant mice contained 14-HRR and AR, but the retinoic acid concentration did not increase compared to controls. In summary, the retro-retinoids 14-HRR and AR are weaker teratogens than retinol. The low teratogenicity observed might be due to the facts that 14-HRR and AR do not contain the terminal carboxylic group involved in binding and activation of the retinoic acid nuclear receptors and they are not metabolized to acidic retinoids.

Removal of LIF (leukemia inhibitory factor) results in increased vitamin A (retinol) metabolism to 4-oxoretinol in embryonic stem cells.

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological ( approximately 1 microM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced (>15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.

Anhydroretinol induces oxidative stress and cell death.

The retro-retinoid anhydroretinol (AR), a physiological metabolite of retinol (vitamin A), induces cell death in multiple in vitro systems. AR-induced cell death is blocked by retinol and its metabolite 14-hydroxy-4,14-retro-retinol. AR has been shown also to prevent mammary cancer induced by N-methyl-N-nitrosourea in rats. We report that AR kills cells by generating reactive oxygen species. Direct measurements show that the addition of AR to lymphoblastoid cells increases the intracellular oxidative stress in a time- and dose-dependent manner. Furthermore, the amount of induced oxidative stress directly correlates with the number of dying cells. The addition of retinol, 14-hydroxy-4,14-retro-retinol, or the antioxidant, alpha-tocopherol (vitamin E), decreases AR-induced oxidative stress and proportionally reduces AR-induced cell death. In contrast, pretreatment with caspase inhibitors, known to inhibit apoptosis, has no effect on AR-induced cell death. This is the first demonstration of cellular reactive oxygen species production by a natural retinoid.

4-Oxoretinol, a metabolite of retinol in the human promyelocytic leukemia cell line NB4, induces cell growth arrest and granulocytic differentiation.

All-trans-retinoic acid (RA) is used as a differentiation therapy for acute promyelocytic leukemia. Patients can become resistant to RA, and this resistance is thought to be mediated in part by an increase in the rate of RA metabolism. We have characterized the metabolism of all-trans-retinol (ROL; vitamin A) in NB4 cells, which are human promyelocytic leukemia cells. NB4 cells metabolize ROL into a variety of compounds, including all-trans-4-hydroxyretinol, all-trans-4-oxoretinol (4-oxoROL), 14-hydroxy-4,14-retro-retinol, anhydroretinol, and several ROL esters. No metabolism of ROL to RA or to RA derivatives in NB4 cells was detected. The rate of ROL metabolism increased after cell differentiation; in a 24-h period, differentiated cells metabolized 2-fold more ROL than did undifferentiated cells. The major difference in the ROL metabolism pattern between undifferentiated and differentiated cells was an approximately 10-fold increase in the production of all-trans-4-hydroxyretinol and 4-oxoROL in differentiated cells. Furthermore, exogenously added 4-oxoROL was capable of eliciting NB4 cell differentiation, as measured by growth inhibition, nitroblue tetrazolium reduction, nuclear body relocalization of PML, and surface expression of CD11b. In addition, 4-oxoROL synergized with IFN-gamma in the promotion of NB4 cell growth arrest. Following treatment of NB4 cells with 4-oxoROL to induce differentiation, the production of 4-oxoROL from ROL was observed; this indicated that 4-oxoROL induces its own synthesis in NB4 cells. In addition, 48 h after the administration of 1 microM 4-oxoROL, NB4 cells maintained a high intracellular concentration (17 microM) of 4-oxoROL. These unique properties of 4-oxoROL may provide advantages over RA in the treatment of promyelocytic leukemia cells because it may be possible to maintain cytodifferentiating concentrations of 4-oxoROL in the cells for extended periods of time.

Human breast cancer cells and normal mammary epithelial cells: retinol metabolism and growth inhibition by the retinol metabolite 4-oxoretinol.

To understand the signaling and growth-inhibitory effects of retinoids, we have examined the metabolism of [3H]retinol in a number of estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) human breast cancer cell lines. We have also assayed the metabolism of [3H]retinol in normal human mammary epithelial cells. The ER+ breast cancer cell lines MCF-7 and T47D produce [3H]4-oxoretinol from [3H]retinol; the production of [3H]4-oxoretinol is increased by initial culture in the presence of nonradiolabeled retinoic acid (RA) or N-(4-hydroxyphenyl)retinamide, indicating that these drugs enhance [3H]retinol metabolism to [3H]4-oxoretinol. No metabolism of [3H]retinol to [3H]RA in these ER+ tumor lines was detected. ER- breast cancer lines MDA-MB-231, MDA-MB-468, and BT20 do not metabolize [3H]retinol to [3H]4-oxoretinol. In the ER- tumor lines, most of the [3H]retinol remains unmetabolized during the 24-h culture period; MDA-MB-468 and BT20 metabolize some [3H]retinol to [3H]RA. Unlike the majority of the tumor lines, the normal human breast epithelial cell strains AD074 and MCF10A rapidly metabolize [3H]retinol to [3H]retinyl esters. No detectable [3H]RA is produced from [3H]retinol in AD074 and MCF10A cells. Thus, the normal breast epithelial strains, the ER+ tumor lines and the ER- tumor lines differ greatly in their pathways of [3H]retinol metabolism. The levels of cellular retinol binding protein-I mRNA expression are not correlated with the levels or types of various retinol metabolites. Whereas the normal breast epithelial cells and the ER+ tumor lines are growth inhibited by RA, N-(4-hydroxyphenyl)retinamide, and 4-oxoretinol, only the 4-oxoretinol is growth inhibitory in the ER- tumor lines. The cellular retinoic acid-binding protein II mRNA levels are not correlated with the growth inhibition by RA or 4-oxoretinol in the normal and tumor lines.

Vitamin A in serum is a survival factor for fibroblasts.

Murine 3T3 cells arrest in a quiescent, nondividing state when transferred into medium containing little or no serum. Within the first day after transfer, fibroblasts can be activated to proliferate by platelet-derived growth factor (PDGF) alone; cells starved longer than 1 day, however, are activated only by serum. We demonstrate that endogenous vitamin A (retinol) or retinol supplied by serum prevents cell death and that retinol, in combination with PDGF, can fully replace serum in activating cells starved longer than 1 day. The physiological retinol derivative 14-hydroxy-4, 14-retro-retinol, but not retinoic acid, can replace retinol in rescuing or activating 3T3 cells. Anhydroretinol, another physiological retinol metabolite that acts as a competitive antagonist of retinol, blocks cell activation by serum, indicating that retinol is a necessary component of serum. It previously has been proposed that activation of 3T3 cells requires two factors in serum, an activation factor shown to be PDGF and an unidentified survival factor. We report that retinol is the survival factor in serum.

Retro-retinoids in regulated cell growth and death.

Vitamin A serves as a prohormone from which three classes of active metabolites are derived: the aldehydes, the carboxylic acids, and the retro-retinoids. Although these three classes are united under the rubric of signal transduction, they act by different molecular mechanisms: the 11-cis-retinaldehydes combine with opsin to form the universal visual pigments and the retinoic acids form ligands for transcription factors, whereas the retro-retinoids, as shown here, intersect with signal transduction at a cytoplasmic or membrane site. The retro-retinoid, anhydroretinol (AR), has long been known to act as a growth inhibitor in lymphocytes, whereas 14-hydroxy-4,14-retro-retinol (14-HRR) is required for normal lymphocyte proliferation. A mutually reversible relationship exists between these two retro-retinoids as one can reverse the effects of the other when given in pharmacological doses. The common explanation for reversible inhibition is competition for a shared receptor. We now provide evidence that when AR is given to T cells unmitigated by 14-HRR, rapid cell death can occur. The circumstances are closely related to nonclassical forms of apoptosis: within 2 h of AR administration the T cells undergo widespread morphological changes, notably surface blebbing and ballooning and, inevitably, bursting. In contrast, nuclear changes are comparatively mild, as indicated by absence of chromatin condensation and overt DNA cleavage to discrete nucleosomal fragments, although DNA nicks are readily discernible by terminal deoxynucleotidyl transferase assay. What further distinguishes the AR-induced form of apoptosis from classical ones is a lack of requirements of messenger RNA and protein synthesis, suggesting that the events leading to cell death are primarily initiated and play themselves out in the cytoplasm. This view is further reinforced by the finding that herbimycin A can prevent the onset of programmed cell death. The importance of our findings is that they strongly suggest a second messenger role for vitamin A metabolites in the cytoplasmic realm that has not been seen previously. These findings are entirely compatible with a general notion that in a cell requiring multiple coordinated signals for survival, the provision of an unbalanced signal can initiate programmed cell death. Collectively, our data also challenge the paradigm that retinoids (outside vision) solely mediate their function via the steroid/ retinoic acid receptor family of nuclear transcription factors. Instead, a mode of action in the cytoplasmic realm akin to one attributed to other small lipophilic second messenger molecules, such as diacyl glycerol or ceramide, may apply to retro-retinoids.

Extraction of human epidermis treated with retinol yields retro-retinoids in addition to free retinol and retinyl esters.

Vitamin A, all-trans-retinol, is metabolized to retinoic acid in vivo by a tightly controlled two-step conversion. Retinoic acid then binds to nuclear receptors and modulates cellular proliferation and differentiation. Because only a small fraction of retinol applied topically can be metabolized to retinoic acid, alternative pathways of retinol metabolism in skin were investigated. Retinol (0.4%) was applied to adult human skin under occlusion for 6 h to 4 d. The conversion of retinol into various metabolites such as 14-hydroxy-4,14-retro-retinol, anhydroretinol, 4-oxo-retinol, retinyl esters, and retinyl glucuronides was investigated. The level of 14-hydroxy-retro-retinol was increased from undetectable at time 0 to 326 ng/g wet weight of tissue at 6 h (6% of the retinol level) and maintained approximately the same concentration at 24 h to 409 ng/g wet weight (1.9% of the retinol level); it decreased to 48 ng/g wet weight of tissue (12% of its maximum level) by 4 d. Anhydroretinol was undetectable at time 0, increased only slightly at 6 h, and remained at the same level. We did not detect 4-oxo-retinol. Because 14-hydroxy-retro-retinol was found in the retinol-treated areas, its effects on epidermis were compared with those of retinol. Topical application of trans-retinol (0.3%) significantly increased both epidermal thickness and cellular retinoic acid binding protein II mRNA, whereas 14-hydroxy-4,14-retro-retinol (0.3%) did not increase either of these well-characterized cutaneous retinoid responses. Retinol, when applied topically in pharmacologic doses to human epidermis, remained as free retinol, was metabolized primarily to retinol ester, and was metabolized to a lesser extent to retro-retinoids and didehydroretinol.

Novel retinoic acid receptor ligands in Xenopus embryos.

Retinoids are a large family of natural and synthetic compounds related to vitamin A that have pleiotropic effects on body physiology, reproduction, immunity, and embryonic development. The diverse activities of retinoids are primarily mediated by two families of nuclear retinoic acid receptors, the RARs and RXRs. Retinoic acids are thought to be the only natural ligands for these receptors and are widely assumed to be the active principle of vitamin A. However, during an unbiased, bioactivity-guided fractionation of Xenopus embryos, we were unable to detect significant levels of all-trans or 9-cis retinoic acids. Instead, we found that the major bioactive retinoid in the Xenopus egg and early embryo is 4-oxoretinaldehyde, which is capable of binding to and transactivating RARs. In addition to its inherent activity, 4-oxoretinaldehyde appears to be a metabolic precursor of two other RAR ligands, 4-oxoretinoic acid and 4-oxoretinol. The remarkable increase in activity of retinaldehyde and retinol as a consequence of 4-oxo derivatization suggests that this metabolic step could serve a critical regulatory function during embryogenesis.

4-Oxoretinol, a new natural ligand and transactivator of the retinoic acid receptors.

All-trans-retinoic acid (at-RA) induces cell differentiation in a wide variety of cell types, including F9 embryonic teratocarcinoma cells, and can influence axial pattern formation during embryonic development. We now identify a novel retinoid synthetic pathway in differentiating F9 cells that results in the intracellular production of 4-oxoretinol (4-oxo-ROL) from retinol (vitamin A). Approximately 10-15% of the total retinol in the culture is metabolized to 4-hydroxyretinol and 4-oxo-ROL by the at-RA-treated, differentiating F9 cells over an 18-hr period, but no detectable metabolism of all-trans-retinol to at-RA or 9-cis-retinoic acid is observed in these cells. Remarkably, we show that 4-oxo-ROL can bind and activate transcription of the retinoic acid receptors whereas all-trans-retinol shows neither activity. Low doses of 4-oxo-ROL (e.g., 10(-9) or 10(-10 M) can activate the retinoic acid receptors even though, unlike at-RA, 4-oxo-ROL does not contain an acid moiety at the carbon 15 position. 4-oxo-ROL does not bind or transcriptionally activate the retinoid X receptors. Treatment of F9 cells with 4-oxo-ROL induces differentiation without conversion to the acid and 4-oxo-ROL is active in causing axial truncation when administered to Xenopus embryos at the blastula stage. Thus, 4-oxo-ROL is a natural, biologically active retinoid that is present in differentiated F9 cells. Our data suggest that 4-oxo-ROL may be a novel signaling molecule and regulator of cell differentiation.

Spectral tuning in bacteriorhodopsin in the absence of counterion and coplanarization effects.

The basis for wavelength regulation in bacteriorhodopsin (BR) and retinylidene proteins in general has been studied for decades but is still only partially understood. Here we report the preparation and spectroscopic characterization of BR analogs aimed at investigating the existence of spectral tuning mechanisms other than the two widely accepted mechanisms, weakened counterion interactions and ring/chain coplanarization. We synthesized two novel retinal analogs containing a saturated 13-14 bond, which interrupts the interaction of the protein counterions with the chromophore conjugation system. Furthermore, one of the analogs has a planar polyene system so that the contribution to the red shift of BR by retinal ring/chain coplanarization is also absent. We incorporated these analogs into bacterioopsin and discovered a sizable amount of red shift, which can be accounted for by interactions between the polar or polarizable groups of the protein and the retinal polyene chain. Our results suggest that the wavelength regulation in BR is achieved by synergistic chromophore/protein interactions including ring/chain coplanarization, excited state stabilization by polar or polarizable protein side chains located along the polyene chain, and weakened counterion interactions near the Schiff base positive charge.

13,14-Dihydroxy-retinol, a new bioactive retinol metabolite.

Deprivation of vitamin A (retinol) leads to reduced potential of B cell proliferation and nearly complete block of T cell activation in vitro. Retinol, which is thought to function as a pro-hormone, is enzymatically converted into intracellular messenger molecules. Thus, 14-hydroxy-retro-retinol (14-HRR) is an intracellular messenger molecule linked to activation and growth regulation of lymphocytes; whereas, anhydroretinol, another natural retro-retinoid, is an antagonist of 14-HRR effects. In this article, we describe the isolation, structure determination, synthesis, and biological properties of a new intracellular retinol derivative, 13,14-dihydroxy-retinol (DHR), which also supports the viability of retinol-deprived lymphocytes. DHR is found in numerous cell lines representing a large cross-section of tissues and animals from insects to mammals. In T lymphocytes the production of DHR and 14-HRR is up-regulated by phorbol ester. DHR is converted to 14-HRR by mild acid treatment, but not by cells; therefore DHR is not a biosynthetic intermediate in the conversion of retinol to 14-HRR. DHR is a distinct end point of retinol metabolism. Although it is linked to cell proliferation, its biological role remains to be determined.

Relief of opsin desensitization and prolonged excitation of rod photoreceptors by 9-desmethylretinal.

The 9-methyl group of 11-cis-retinal plays a crucial role in photoexcitation of the visual pigment rhodopsin. A hydrogen-substituted analogue, 11-cis-9-desmethylretinal, combines with opsin to form a pigment that produces abnormal photoproducts and diminished activation of the GTP-binding protein transducin in vitro. We have measured the formation of this analogue pigment in bleached salamander rods and determined the size and shape of its quantal response. In addition, we have characterized the influence of opsin and newly formed analogue pigment on the quantal response to native porphyropsin. We find that, as 11-cis-9-desmethylretinal combines with opsin in bleached rods, the amplitude of the quantal response from residual native pigment is elevated by approximately 7.5-fold to 0.15 +/- 0.09 pA, a value close to the amplitude of the quantal response before bleach (0.31 +/- 0.10 pA). When activated by light, the new analogue pigment produces a quantal response that is approximately 30-fold smaller and decays approximately 5 times more slowly than that of native pigment in unbleached cells. We conclude that the 9-methyl group of retinal is not critical for conversion of opsin to its nondesensitizing state but that it is critical for the normal processes of activation and deactivation of metarhodopsin that give rise to the quantal response.

Unbleachable rhodopsin with an 11-cis-locked eight-membered ring retinal: the visual transduction process.

Visual transduction occurs through photorhodopsin, the primary photoproduct of rhodopsin, which relaxes to bathorhodopsin and a series of other intermediates until it reaches the metarhodopsin II stage, upon which the enzymatic cascade leading to vision is activated. Despite advances in areas related to visual transduction, the triggering process itself, a key problem in the chemistry of rhodopsin, has remained unsolved. In order to clarify the extent of involvement of the chromophoric excited state versus the 11-cis to trans isomerization, and as an extension of past studies with 11-cis-locked seven-membered ring rhodopsin (Rh7), 11-cis eight- and nine-membered ring retinal analogs, ret8 and ret9, respectively, have been synthesized. The bulkiness of the tetramethylene bridge in ret8 led to numerous unexpected obstacles in attempts to reconstitute a ret8-containing rhodopsin (Rh8) embedded in lipid bilayer membranes. These obstacles were solved by using methylated rhodopsin which gave MeRh8 containing 11-cis-ret8 as its chromophore. MeRh8 exhibited UV-vis and CD spectra very similar to those of native rhodopsin (Rh); furthermore, the quantum efficiency of photorhodopsin formation was comparable to that of Rh.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular signaling activity of synthetic (14R)-, (14S)-, and (14RS)-14-hydroxy-4,14-retro-retinol.

14-Hydroxy-4,14-retro-retinol (14-HRR), first isolated from cultures of lymphoblastoid 5/2 and HeLa cells and characterized by NMR, UV, and CD, is a metabolite of retinol which promotes growth of B lymphocytes in culture and activation of T lymphocytes by antigen receptor-mediated signals. It is also produced by various tested cell lines: fibroblasts, leukemia, and Drosophila cells. 14-HRR is the first bioactive retro-retinoid to be discovered and, after retinal and retinoic acid, is the third intracellular messenger molecule derived from retinol. Physical properties and intracellular signaling activities of synthetic (14R)-HRR, (14S)-HRR, and racemic 14-HRR are described. CD spectra indicate that natural 14-HRR isolated previously was a mixture of enantiomers. B-cell survival and T-cell activation assays performed in the optimal range of (7-1.6) x 10(-7) M surprisingly showed that all 14-HRR compounds exhibit similar activity, with the 14R enantiomer exhibiting slightly higher activity in comparison to the 14S enantiomer. However, because of the semiquantitative nature of the assays, the conclusion as to which enantiomer is more active and which is the true ligand for the target receptor must await characterization of this protein.

Anhydroretinol: a naturally occurring inhibitor of lymphocyte physiology.

Vitamin A (retinol) is an essential cofactor for growth of B lymphocytes in culture and for activation of T lymphocytes by antigen receptor-mediated signals. 14-hydroxy-4,14-retro-retinol (14-HRR) a metabolite of retinol, has been implicated as the intracellular mediator of this effect. Anhydroretinol (AR) is a retinol derivative with retro structure produced in activated human B lymphocytes and the insect cell lines SF 21 and Schneider S2. AR reversibly inhibits retinol- and 14-HRR-dependent effects and blocks B lymphocyte proliferation as well as activation of resting T lymphocytes. The intracellular signaling pathway blocked by AR in T cell activation is distinct from the calcineurin/interleukin 2 pathway inhibitable by cyclosporine A or FK-506.

Comparative study of phototactic and photophobic receptor chromophore properties in Chlamydomonas reinhardtii.

The motile, unicellular, eukaryotic alga Chlamydomonas reinhardtii exhibits two distinct behavioral reactions to light stimuli, phototaxis and the photophobic response. Both are mediated by retinal-containing receptors. This paper focuses on a direct comparison of the two photoresponses and the chromophore requirements for their photoreceptor(s). Using computerized motion analysis assays for phototaxis and photophobic responses by the same populations of cells, we measured the ability of various isomers and analogues of retinal to reconstitute photobehavior in the pigment-deficient mutant FN68. The results indicate that photophobic and phototaxis responses each require chromophores with an all-trans polyene chain configuration, planar ionone ring/polyene chain conformation, and the ability to isomerize around the retinal C13-C14 double bond. One difference between the two behaviors is that the photophobic response becomes highly desensitized after light stimuli to which the phototaxis response does not become desensitized, indicating the existence of at least one distinct step in the photophobic response pathway. A second difference is that the retinal regeneration of the photophobic response but not of phototaxis is inhibited by a 5-membered ring 13-trans-locked analogue. While showing close similarity in the chromophore structural requirements of the two behaviors, the results indicate that differences exist between the two responses at the level of their photoreceptor proteins and/or in their transduction processes.

Photoisomerization mechanism of the rhodopsin chromophore: picosecond photolysis of pigment containing 11-cis-locked eight-membered ring retinal.

The primary photochemical event in rhodopsin is an 11-cis to 11-trans photoisomerization of its retinylidene chromophore to form the primary intermediate photorhodopsin. Earlier picosecond studies have shown that no intermediate is formed when the retinal 11-ene is fixed through a bridging five-membered ring, whereas a photorhodopsin-like intermediate is formed when it is fixed through a flexible seven-membered ring. Results from a rhodopsin analog formed from a retinal with locked 11-ene structure through the more flexible eight-membered ring (Ret8) are described. Incubation of bovine opsin with Ret8 formed two pigments absorbing at 425 nm (P425) and 500 nm (P500). P425, however, is an artifact because it formed from thermally denatured opsin or other proteins and Ret8. Excitation of P500 with a picosecond green pulse led to formation of two intermediates corresponding to photo- and bathorhodopsins. These results demonstrate that an appearance of early intermediates is dependent on the flexibility of the 11-ene and that the photoisomerization of P500 proceeds by stepwise changes of chromophore-protein interaction, which in turn leads to a relaxation of the highly twisted all-trans-retinylidene chromophore in photorhodopsin.

Diversion of the sign of phototaxis in a Chlamydomonas reinhardtii mutant incorporated with retinal and its analogs.

The blind mutant FN68 of the unicellular flagellate green alga Chlamydomonas reinhardtii is negatively phototactic in the presence of the native chromophore all-trans retinal. In contrast, analog chromophores such as a ring-acyclic retinal and those in which trans/cis isomerization about the C11 = C12 double bond was blocked induced predominantly positive phototaxis in the same strain under the same experimental conditions. These observations can be interpreted by assuming that the negative and the positive phototaxis is mediated distinctively by two rhodopsin species which differ in their affinities with the exogenous chromophores. However, a more reasonable explanation, which requires fewer assumptions, is that the sign of phototaxis depends on a delay in intracellular photosignal transduction. This novel view was deduced directly from the widely accepted hypothesis [1980, Microbiol. Rev. 44, 572-630] on phototaxis mechanisms.

Retinal analog restoration of photophobic responses in a blind Chlamydomonas reinhardtii mutant. Evidence for an archaebacterial like chromophore in a eukaryotic rhodopsin.

The strain CC-2359 of the unicellular eukaryotic alga Chlamydomonas reinhardtii originally described as a low pigmentation mutant is found to be devoid of photophobic stop responses to photostimuli over a wide range of light intensities. Photophobic responses of the mutant are restored by exogenous addition of all-trans retinal. We have combined computer-based cell-tracking and motion analysis with retinal isomer and retinal analog reconstitution of CC-2359 to investigate properties of the photophobic response receptor. Most rapid and most complete reconstitution is obtained with all-trans retinal compared to 13-cis, 11-cis, and 9-cis retinal. An analog locked by a carbon bridge in a 6-s-trans conformation reconstitutes whereas the corresponding 6-s-cis locked analog does not. Retinal analogs prevented from isomerization around the 13-14 double bond by a five-membered ring in the polyene chain (locked in either the 13-trans or 13-cis configuration) do not restore the response, but enter the chromophore binding pocket as evidenced by their inhibition of all-trans retinal regeneration of the response. Results of competition experiments between all-trans and each of the 13-locked analogs fit a model in which each chromophore exhibits reversible binding to the photoreceptor apoprotein. A competitive inhibition scheme closely fits the data and permits calculation of apparent dissociation constants for the in vivo reconstitution process of 2.5 x 10(-11) M, 5.2 x 10(-10) M, and 5.4 x 10(-9) M, for all-trans, 13-trans-locked and 13-cis-locked analogs, respectively. The chromophore requirement for the trans configuration and 6-s-trans conformation, and the lack of signaling function from analogs locked at the 13 position, are characteristic of archaebacterial rhodopsins, rather than the previously studied eukaryotic rhodopsins (i.e., visual pigments).