Opsin Research Articles

In vitro differentiation of human amniotic epithelial stem cells into keratinocytes regulated by OPN3.

Human amniotic epithelial stem cells (hAESCs) are regarded as potential alternatives to keratinocytes (KCs) used for skin wound healing. Light is an alternative approach for inducing stem cell differentiation. Opsins (OPNs), a family of light-sensitive, G protein-coupled receptors, play a multitude of light-dependent and light-independent functions in extraocular tissues. However, it remains unclear whether the light sensitivity and function of OPNs are involved in light-induced differentiation of hAESCs to KCs. Herein, we determine the role of OPNs in differentiation of hAESCs into KCs through cell and molecular biology approaches invitro. It is shown that mRNA expression of OPN3 in the amniotic membrane and hAESCs was higher than the other four primary OPNs by RT-qPCR analysis. Changes in OPN3 gene expression had a significant impact on cell proliferation, stemness and differentiation capability of hAESCs. Furthermore, we found a significant upregulation of OPN3, KRT5 and KRT14 with hAESCs treated at 3 × 33 J/cm2 irradiation from blue-light LED. Taken together, these results suggest that OPN3 acts as a positive regulator of differentiation of hAESCs into KCs. This study provides a novel insight into photosensitive OPNs associated with photobiomodulation(PBM)-induced differentiation in stem cells.

616 Ultraviolet A mediates the keratinocytes supranuclear melanin cap formation via opsin 3

Background: the human keratinocytes supranuclear melanin cap acts as a microparasol to protect the nucleus from ultraviolet (UV) induced DNA damage. UV induced cytoplasmic dynein expression mediates supranuclear melanin cap formation. However, the molecular mechanism of keratinocytes responding to UV irradiation and mediating melanin cap formation remains unclear. Opsins(OPNs) belong to the G protein-coupled receptors superfamily and are photosensitive receptor proteins mediating phototransduction by translation of absorbed photons to cellular responses.

Identification of novel bladder sensory GPCRs.

Sensory GPCRs such as olfactory receptors (ORs), taste receptors (TRs), and opsins (OPNs) are now known to play important physiological roles beyond their traditional sensory organs. Here, we systematically investigate the expression of sensory GPCRs in the urinary bladder for the first time. We find that the murine bladder expresses 16 ORs, 7 TRs, and 3 OPNs. We additionally explore the ectopic expression of these GPCRs in tissues beyond the bladder, as well as the localization within the bladder. In future work, understanding the functional roles of these bladder sensory GPCRs may shed light on novel mechanisms which modulate bladder function in health and disease.

Application and research progress of optogenetic technology in retinal diseases

Optogenetics is a technique combining optics and the power of light with genetics; it uses light-mediated protein-protein interactions to control the open/closed state of channels or the activation/inactivation states of signaling components within live cells.Recently developed optogenetic tools offer exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution.The eye is a light-bioelectric conversion system.Compared with diseases of other organs, there are great advantages to apply optogenetics to the treatment of ophthalmic conditions, as new optical genetic tools are rapidly emerging.This article will focus on the main methods of optogenetic control of cells and the current status and outlook of its application in retinal diseases. Key words: Optogenetics; Opsins; Retinal diseases

Degeneration of short-wavelength cone cells in rd12 mice

Background Retinitis pigmentosa (RP) is one of the causes of congenital blindness.It is well known that the degeneration process of rod cells is difficult to detect in RP.Retinal degeneration 12 (rd12) mice is a new, spontaneously arising mouse model for human Leber congenital amaurosis (LCA), and it is helpful for us to explore the pathogenesis and determine the treating target of RP. Objective This study was to investigate the natural disease process of short-length sensitive cone cells in rd12 mice, a LCA Rpe65rd12 (B6[A]-Rpe65rd12/J) mouse. Methods The rd12 mice at postnatal (P) 14, P21, P35 and P90 were selected (5 mice for each), and the wild-type C57BL/6J (B6) mice with matched ages were included as controls.Photopic full-field electroretinogram (ERG) was recorded with Roland Q450SC UV visual physiology instrument.Cone response was recorded using single white light-emitting diode (LED) stimulation with the flash intensity of 1.00 cds/m2 and 1.96 cds/m2, and short wave-length sensitive cone response was recorded using ultraviolet light ([363±6]nm) stimulation with the flash intensity of 2.0 mWs/m2 and 3.0 mW/m2.The mice were sacrificed and retinal whole-mounts were prepared.The distribution and number of cone cells and UV-sensitive cone cells were detected by FITC-peanut agglutinin (FITC-PNA) and Cy3 immunofluorescence stainning, respectively. Results In P14 rd12 mice, the ERG responses of overall cone cells presented the negative waveform and the latency was delayed, and UV-sensitive cone response was unrecordable.The b-wave amplitude of overall cone cells reduced by 75% in P21 rd12 mice compared with wild-type B6 mice, and the mean latency of b-wave in the P21 rd12 mice was significantly longer than that in the wild-type B6 mice ([102.80±11.39]ms vs.[43.40±5.60]ms) (t=-8.106, P=0.001). The mean b-wave amplitudes of UV-sensitive cone cells were (59.60±36.00), (82.40±12.22) and (68.43±17.63)μV in the wild-type B6 mice, and those in the rd12 mice were unrecordable.Immunofluorescence showed that a lager number of cone cells with green fluorescence were seen, and the expression of opsin with red fluorescence was displayed in the UV-sensitive cone cells of nasal lateral on retinal ventral side in P14 wild-type B6 mice; while only a few opsin positive-response cells were seen in P14, P21 and P35 rd12 mice. Conclusions In rd12 mice, the functional abnormality and quantitative reduction of cone cells appear in the early postnatal days, and the loss of UV-sensitive cone cells is earlier and more obvious. Key words: Retinal degeneration/congenital; Eye proteins/metabolism; Photoreceptor/pathology; Electroretinography; Fluorescent antibody technique; Leber congenital amaurosis; Mice, inbred C57BL; Disease models, animal

621 Melanocytes sense blue-light and regulate the pigmentation through the Opsin 3

621 Melanocytes sense blue-light and regulate the pigmentation through the Opsin 3

530 Soy isoflavones and nicotinamide down-regulate pro-inflammatory mediators in vitro

530 Soy isoflavones and nicotinamide down-regulate pro-inflammatory mediators in vitro R Di Caprio, S Lembo, A Balato, F Gasparri and G Monfrecola 1 Deparment of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy, 2 Department of Medicine and Surgery, University of Salerno, Salerno, Italy, 3 Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy and 4 Department of Pharmacy (DIFARMA), University of Salerno, Salerno, Italy The aim of this study was to evaluate, in vitro, the anti-inflammatory and antioxidant properties of soy isoflavones and nicotinamide (NCT), singularly or in combination, in UVBirradiated keratinocytes. Soy isoflavones were used in two different forms: ISOSOY and Lo ISO-3s. The first, is a standardized glycine soy extract titrated to 90% in isoflavone aglycones (with genistein:daidzein1⁄41:4), soluble in dimethylsulfoxide. The second, consists in microencapsulated soy extracts, enriched in daidzein and genistein, obtained by spray-drying technique using a sodium-carboxymethylcellulose based matrix, soluble in water. Immortalized human keratinocytes were pre-incubated for 2h with ISOSOYand NCTor with Lo ISO3s and NCT, singularly or in combination, and irradiated with UVB 60 mJ/cm. Substances toxicity was assessed through cell viability and their possible anti-inflammatory effect was explored analyzing IL-6, TNF-a and COX-2 gene expression (qRT-PCR) 24h after UVB irradiation. None of the tested substances resulted toxic to cells. All of them showed anti-inflammatory properties alone or in combination with NCT. In detail, Lo ISO-3s was more effective than ISOSOY in down-regulating IL-6 and COX-2 increases (p<0.01), while ISOSOY was more effective than Lo ISO-3s in down-regulating TNF-a increases (p<0.01). A part from TNF-a, Lo ISO-3s was more effective than NCT in reducing UVB-induced inflammation, while ISOSOY, generally, resulted less effective. In conclusion, soy isoflavones and NCT, used alone or in combination, have demonstrated a potential immuno-modulatory and cytoprotective effect representing an interesting option to improve or prevent UV-induced/aggravated clinical conditions. 531 Human skin mast cells express photoreceptors H Siiskonen, S Buscone, I Castellano Pellicena, A Smorodchenko, NE Uzunbajakava, NV Botchkareva, M Maurer and J Scheffel 1 Faculty of Life Sciences, Centre for Skin Sciences, Bradford, United Kingdom, 2 Dermatology, Kuopio University Hospital, Kuopio, Finland, 3 Department of Dermatology and Allergy, Charite Universitatsmedizin Berlin, Berlin, Germany and 4 Philips Research Eindhoven, Eindhoven, Netherlands Circadian clocks critically influence mast cell (MC) functions and drive the daily rhythms in IgE/MCemediated allergic reactions. In the skin, these clocks may be regulated by photoreceptors (PRs) such as cryptochromes (CRY) and opsins (OPN), which have recently been shown to be expressed in keratinocytes. Whether skin MCs express PRs has not been investigated yet. Therefore, we studied the expression of selected PRs in human MCs using qRT-PCR. To this end, we used freshly isolated cutaneous MCs from breast (n1⁄43), eyelid (n1⁄41) and abdominal skin (n1⁄41); cultured MCs from breast (n1⁄42) and foreskin (n1⁄42); cultured CD34-positive peripheral blood stem cell-derived MCs (PSCMCs); and LAD2-MCs. We identified that freshly isolated MCs from breast skin, cultured MCs from breastand foreskin and LAD2-MCs express CRY1, OPN1 medium-wave-length, OPN2 and OPN3. Expression of CRY1, OPN1 medium-wave-length and OPN3 was also detected in freshly isolated MCs from eyelid skin. Of the PRs assessed, CRY1 was the most prominently expressed, and we also found CRY1 expression at the protein level in cultured MCs from breast skin and foreskin and from freshly isolated MCs from breast skin. The expression levels of PRs varied between different donors and skin areas. MCs freshly isolated from abdominal skin and PSCMCs were negative for the investigated PRs, suggesting that expression of CRYs and OPNs might be connected to MC differentiation and functionality. Our results show, for the first time, that human skin MCs express several PRs. We will next investigate a potential role of these PRs in MCs by studying their involvement in circadian oscillation of MC function and in MCmediated skin responses.

The application of optogenetics in the treatment of retinal degeneration disease

Optogenetics is a novel technique which combines optics with genetics. Using genetic means, a selected opsin protein is ectopically expressed in target neurons, which are then stimulated by light to moderate the neuronal circuit, as a consequence to regulate the animal's behaviors. Retinal degeneration like retinitis pigmentosa and aged macular degeneration causes visual impairment and eventual blindness. Optogenetics techniques have opened the door to creating artificial photoreceptors in the remaining retinal circuits of retinal degeneration retinas via gene therapy. However, there are still limitations in optogenetics technique, for example, potential risk in virus infection, the choice of target cells and the low visual resolution of the experiment animal. It has been reported that vision was successfully restored to a certain extent in animal model using optogenetics technique. With higher photosensitivity of opsin protein, longer activation kinetics and higher transfection efficiency of virus vector, optogenetics techniques' application in ophthalmology will be improved. Key words: Retinal degeneration/therapy; Opsins; Genetic techniques; Review

Anti-aging effect of simvastatin and telmisartan on retinas and its mechanism in rats

Background Statins has prominent roles in regulating lipids, anti-inflammation, autoxidation and protecting vascular endothelial cells.Sartans can promote cell growth and the expression of cytokines.Since the pleiotropic effects of statins and sartans on a variety of cell types, it is inferred that the two medicines can delay retinal aging. Objective This study was to explore the anti-aging effect of simvastatin and telmisartan on the physiological aging of retina. Methods Sixty-six three-month-old healthy SD rats were selected in this study, and 6 of them served as the youth group and the right eyeballs were immediately enucleated.The other rats were raised until 9-month-old in the same conditions and then randomly divided into the simvastatin group, telmisartan group and the control group with 20 rats for each group.The simvastatin of 5 mg/kg and telmisartan of 8 mg/kg were given by intragastric administration once a day in the simvastatin group and the telmisartan group until 17-month-old, and the equal amount of normal saline was used in the control group in the same way.The number of survival rats was 12 in the simvastatin group, 10 in the telmisartan group and 8 in the control group.The right eyes were enucleated after heart perfusion of 4% paraformaldehyde solution for the preparation of retinal paraffin sections.Retinal thickness was measured by pathological examination, and the expressions of the retinal neuron markers, including Thy-1, protein kinase C-α (PKC-α), opsin and rhodopsin, were detected by immunofluorescence technique to evaluate the morphology of retinal ganglion cells (RGCs), bipolar cells as well as the thickness of the outer segment of photoreceptors. Results The retinal structure was clear in the rats of the youth group.However, the RGCs arrangement and inner segment (IS) and outer segment (OS) structure were abnormal in the simvastatin group, the telmisartan group and the control group.Compared with the rats of the youth group, the thickness of outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and the total thickness of the aging rats were decreased, and the IS/OS thickness was increased in the simvastatin group and the telmisartan group (all at P<0.01). Thy-1 stain showed that the number of RGCs was reduced in the simvastatin group, telmisartan group and the control group compared with the youth group, and that in the simvastatin group was increased in comparison with the control group (all at P<0.01). PKC-α stain exhibited that the density of bipolar cells was increased but the axon terminal bouton was declined in the simvastatin group, telmisartan group and the control group compared with the youth group, and the axon terminal bouton was declined in the simvastatin group compared with the youth group and the control group (all at P=0.000). Opsin and rhodopsin stains displayed that the OS thickness was increased in the simvastatin group, telmisartan group and the control group compared with the youth group, and that in the telmisartan group was reduced in comparison with the control group (all at P<0.01). Conclusions As SD rat aging, retinal thickness is gradually attenuated and the number of RGCs is gradually declined.Although the density of bipolar cells seem to be unchanged, their synaptic connections are decreased and the OS is thicken.Simvastatin and telmisartan can delay retinal senescence by protecting retinal neurons against aging and thinning thickened OS. Key words: Aging/physiology; Retina/physiopathology; Aged; Eye proteins/drug effect; Neurons/structure; Statins; Sartans; Animal, rats

The distribution and degeneration pattern of the cone opsins in rd11 mice

Background The retinal degeneration 11 (rd11) mouse is a newly discovered naturally occurring recessive animal model with lysophosphatidylcholine acyltransferase 1 (Lpcatl) mutation.Previous studies showed that the photoreceptor cells are characterized by typical rod-cone degeneration pattern in rd1 1 mice,while cone degeneration pattern in rd11 mice is unclcar.Objective Using immunofluorescence staining techniques with retinal wholemount,we aim to clarify the degeneration patterns of cone-function related M-opsin or S-opsin in different ages of rd1 1 mice.Methods A total of thirty rd1 1 and C57BL/6J mice at postnatal (P) day 14,28,42 (five in each age group) were sacrificed and retinal wholemounts were prepared.Immunohistochemistry was performed to identify the expression of M-opsin or S-opsin in retinal wholemounts,which were photographed with a fluorescent microscope.Cone opsins were compared between rd1 1 retinas and age-matched normal C57BL/6J retinas by manually counting the opsin positive cone cells in different quadrants of the retinas.Results The number of M-opsin or S-opsin positive fluorescent dots in each quadrant was similar at all ages of normal C57BL/6J retina.M-opsin positive fluorescent dots in dorsal/temporal,ventral/temporal,dorsal/nasal and ventral/nasal quadrants of rdl 1 retina at P28 were (414±32),(300± 8),(324 ± 22) and (250± 20)/0.037 mm2,which were lower than the age-matched normal C57BL/6J mice (t =4.114,15.225,7.505,17.990,all at P＜0.05).At the same time the S-opsin positive fluorescent dots in P28 rd11 were (8 ±4),(175 ± 16),(74 ± 13) and (315 ±20)/0.037 mm2,with significant decrease in comparison with those in the age-matched normal C57BL/6J mice (t =8.555,17.076,21.637,13.498,all at P＜0.05).With the development of retinal degeneration in rd11 mice,the M-opsin degeneration spread from central to ventral,nasal and then to temporal and dorsal peripheral retina;and the S-opsin loss started from dorsal/temporal to ventral/nasal retina.Conclusions Most of the M-opsin and S-opsins,especially the S-opsins in rd11 mice,degenerate in 6 weeks.Retinal wholemount and cone opsin immunofluorescent staining provide a useful tool to show the cone degeneration pattern and to evaluate the therapeutic efficiency in ongoing gene therapy study. Key words: rd11 Mouse/spontaneous retinal degeneration; Cone; Opsin; Retinal wholemount; Immunofluoreseence staining

各波長LEDに対するトビイロウンカの誘引行動と複眼構造およびオプシン遺伝子

イネ害虫トビイロウンカの光応答とそれに関連する分子に関する基礎的な知見を得るために，フォトン数を合わせられるLEDを用いての誘引実験，オプシン遺伝子同定および複眼の構造解析を行った．暗箱内での粘着板を用いた実験では，一方向からの光照射の場合は365–735 nmの広い波長範囲でトビイロウンカの誘引が見られた．また，両方向から等フォトンの光を照射した場合，選好性は 365 nm＝385 nm＞470 nm＝525 nm＞白色LED＝590 nm＞660 nm＞735 nm＞850 nm の順に有意に高かった．トビイロウンカ複眼の分光感度は520 nmに高いピークと360 nm にそれより低いピークが見られ，660 nm以上の長波長に対しての光受容感度は非常に低かった．トビイロウンカの光受容遺伝子は，長波長オプシン1種とUVオプシン遺伝子2種が見つかった．長波長オプシンは複眼の全体と単眼で，UVオプシン1は触角周縁部の下側で，UVオプシン2は複眼の全体で発現していた．ウンカ複眼の個眼は少なくとも8個の視細胞からなっていた．

Expression of Deep-brain Photoreceptor, Vertebrate Ancient Long Opsin, in the Japanese Medaka

Expression of Deep-brain Photoreceptor, Vertebrate Ancient Long Opsin, in the Japanese Medaka

Influence of long-time illumination of monochromatic light on density of cones and opsin expression in guinea pig

Background The visual system of animal have to optimally adjust in various environmental conditions in order to obtain stable and effective visual funetion.However,the color vision system of animals which encounter uncertainty of spectral signals should be plastic.Whether the densities of various cones and expression of opsins change with long-time spectral deprivation is unclear.Objective This study was to investigate the changes of cone density as well as the expression of corresponding opsin and mRNA following the long-term illumination of monochromatic light.Methods Thirty 3-day-old guinea pigs were randomized into 3 groups and exposed tO the 530 nm green light,400 nm purple light and white light for consecutive 8 weeks respectively.The flat-mounted retinal sample was prepared and divided into dorsal zone,ventral zone and mixed zone anatomically according to the distribution of difierent light-sensitive cone.The changes in density of cone cells sensitivited to different colored light were detected by single-1abel or double-label immunocytochemistry.The levels of opsin and its mRNA were determined using Western-blot and real-time PCR respectively.Results The density of green-sensitivity cones was significantly different in the dorsal zone of retina among green light group,purple light group and white light group (F=234.28,P 0.05).The density of coexpression of the mixed cone cells was increased in green light group(q=157.55,P 0.05；F=3.27,P>0.05).Conclusion After the exposure of long-time monochromatic light illumination,monochromatic cones density and its opsin in guinea pig occur the corresponding alteration to gain good spatial vision as a compensatory reaction.These outcomes imply that there is some plasticity during the development of color vision.The increase of green-sensitive cones might be from the differentiation of coexpression cones in transition region. Key words: Color vision; Cone; Opsin; Green-sensitive cone; Purple-sensitive cone

The effects of varied antigen retrieval methods on immunohistochemistry of functional proteins in retinal paraffin section

Background Antigen retrieval method is the key of improving the successful rate of immunohistochemical assay in paraffin sections.To study an available method of antigen retrieval is a goal to achieve both good immunochemistry result and preserving retinal proteins.ObjectiveThe aim of present study is to investigate tyrosin digestion,high-temperature heat pressure,water bath heating and microwave repair in antigen retrieval for retinal tissues.MethodsRetinal tissue was isolated and obtained from clean Chinchilla rabbits.Four hundreds retinal paraffin sections were prepared.Four kinds of antigen retrieval methods for retinal tissue including tyrosin digestion,high-temperature heat pressure,water bath heating and microwave repair were used respectively.The depigmentated retinal paraffin section without antigen retrieval was used as control.The positive rates of expression of CRALBP,Rhodopsin and opsin proteins were evaluated and compared among four kinds of antigen retrieval methods by immunochemistry.ResultsCRALBP,Rhodopsin and opsin protein was positively expressed in cytoplasm of retinal pigment epithelial cells and the outer segment of photoreceptor respectively.No significant difference was found in the positive expression rates of these three proteins among four kinds of antigen retrieval methods (P＞0.05),but the differences in tissue integrity and background staining were statistically significant (P＜0.01).The structural damage of retina included loss and pucker of scalera,crack of nucleus and abnormal background stain in high-temperature heat pressure method,water bath heating method and microwave retrieval method.However,stable CRALBP,Rhodopsin and opsin protein expression and strain effectiveness,clear background without unspecific staining and integrated tissue were seen in tyrosin digestion method.ConclusionDuring the clinical pathology analysis of retinal tissue,the application of tyrosin digestion in antigen retrieval could obtain a better effectiveness. Key words: antigen retrieval; immunohistochemistry; CRALBP; Rhodopsin; opsin; retina

Functional and Structural Changes in the Retina of Wire-Haired Dachshunds with Early-Onset Cone–Rod Dystrophy

To describe and classify the morphologic changes in a naturally occurring dog model of early-onset cone-rod dystrophy (CRD) and to correlate these with earlier described clinical characteristics of the disease in dogs. Purpose-bred Standard Wire-Haired Dachshunds (SWHDs) derived from a large pedigree of dogs with early-onset CRD were euthanatized at defined ages to characterize morphologic changes in the disease process. Specimens were examined by light microscopy, including morphometric studies, electron microscopy, and immunohistochemistry. Peanut agglutinin (PNA), protein kinase C (PKC), synaptophysin (Syn), rhodopsin (Rho)-63, glial fibrillary acidic protein (GFAP), and short-wavelength cone opsin (OS) were used for immunohistochemical characterization. The photopic cone-system-derived ERG amplitudes were already significantly reduced or nonrecordable in CRD-affected dogs at 5 weeks, the earliest age studied. The outer retina was morphologically most severely affected initially, with a subsequent degeneration of the inner retina. Cone degeneration was more pronounced than rod degeneration in young CRD-affected dogs. There was a marked phenotypic variation based on morphologic findings in the affected dogs. At the earliest time point studied (5-8 weeks) cone photoreceptor and glial cell abnormalities were observed, in accordance with earlier studies based on electrophysiological and clinical findings in which day blindness and abnormal cone ERGs were observed in young affected SWHD puppies. Preliminary genetic studies have indicated an autosomal recessive mode of inheritance for the defect. Through functional and structural characterization, early-onset cone abnormalities were found, consistent with a cone dysplasia at an age when rod structure was normal. Further studies are in progress to identify the gene(s) involved in this retinal disease process. The presently described natural animal model of primary cone dysplasia followed by rod degeneration may provide further insight into the human counterpart. Further studies are needed to ascertain an autosomal recessive mode of inheritance for CRD in the SWHD.

Photopigment coexpression in mammals: comparative and developmental aspects.

In mammals, each cone had been thought to contain only one single type of photopigment. It was not until the early 1990s that photopigment coexpression was reported. In the house mouse, the distribution of color cones shows a characteristic division. Whereas in the upper retinal field the ratio of short wave to middle-to-long wave cones falls in the usual range (1:10), in the ventral retinal field M/L-pigment expression is completely missing. In the transitional zone, numerous dual cones are detectable (spatial coexpression). In other species without retinal division, dual cones appear during development, suggesting that M/L-cones develop from S-cones. Dual elements represent a transitory stage in M/L-cone differentiation that disappear with maturation (transitory coexpression). These two phenomena seem to be mutually exclusive in the species studied so far. In the comparative part of this report the retinal cone distribution of eight rodent species is reported. In two species dual cones appear in adult specimens without retinal division, and dual elements either occupy the dorsal peripheral retina, or make up the entire cone population. This is the first observation proving that all cones of a retina are of dual nature. These species are good models for the study of molecular control of opsin expression and renders them suitable sources of dual cones for investigations on the role and neural connections of this peculiar cone type. In the developmental part, the retinal maturation of other species is examined to test the hypothesis of transitory coexpression. In these species S-pigment expression precedes that of the M/L-pigment, but dual cones are either identified in a small number or they are completely missing from the developing retina. These results exclude a common mechanism for M/L-cone maturation: they either transdifferentiate from S-cones or develop independently.

Light-induced currents in Xenopus oocytes expressing bovine rhodopsin.

1. We have investigated the functioning of bovine rod opsin, which is efficiently synthesized from RNA made by in vitro transcription, following injection into Xenopus oocytes. We found that oocytes expressing the gene for opsin exhibit light-dependent ionic currents only after pigment generation by incubation with 11-cis-retinal. These currents are similar to the endogenous muscarinic acetylcholine (ACh) response of oocytes, but their amplitude is substantially smaller. 2. In order to optimize the conditions for obtaining light-induced currents in RNA-injected oocytes, the native ACh response was examined under several conditions. It was found that elevated external calcium markedly enhances the muscarinic response and that these currents have a non-linear dependence on membrane voltage, increasing substantially with depolarization. 3. Using the optimal conditions for evoking the largest ACh responses, (28 mM [Ca2+]o, 0 mV, omission of serum and Hepes from the media), the light-evoked currents obtained in RNA-injected oocytes were remarkably enhanced, and responses to multiple light stimuli could be obtained. 4. The light response appeared to desensitize, even after long periods of recovery and pigment regeneration. By contrast, the ACh responses continued to appear normal. These results suggest that desensitization of photoresponses expressed in Xenopus oocytes involve changes at early stages of the pathway, resulting in a reduced ability of rhodopsin to couple to the endogenous signalling system.

Linkage of Retinal to Opsin

Linkage of Retinal to Opsin