Satoshi Tamotsu

Photoreception in pineal organs of larval and adult lampreys, Lampetra japonica

SummaryA comparative study of the larval and adult pineal organs, which are sensitive to incident light, was carried out in the river lampreyLampetra japonica, using intracellular recording from the pineal photoreceptors.1.The tissue overlying the larval pineal organ is transparent, whereas that over the adult pineal is translucent. The optical density of this oval pineal window in the adult lamprey was 1.2.2.In order to elucidate the early development of the larval pineal, the ratior of the diameter (μm) of the pineal to the body-length (cm) was measured. The value ofr was 62.5 in a small larva of 2.8 cm, 29.7 in a larger one of 14.3 cm, and 9.3 in an adult of 54 cm body-length.3.The intracellular response to light of the larval pineal was a hyperpolarization, showing fundamentally the same pattern as that of the adult pineal. It was possible to record a typical response even from the pineal of the smallest larva, 2.8 cm in body length, used in this study.4.The intensity-amplitude relationship was analysed after Naka-Rushtons hyperbolic equation. The value ofσ of isolated larval pineals was 0.88 log unit higher than that of adults. The value ofn was larger in larvae, suggesting a sensitive reaction to changing photic stimulus.5.The spectral sensitivity was compared. The peak was at 505 nm in the larva, but 525 nm in the adult. A change of visual pigment in the pineal during metamorphosis is suggested.

Light-modulated subcellular localization of the alpha-subunit of GTP-binding protein Gq in crayfish photoreceptors.

Gq-type GTP-binding protein (Gq) plays an important role in invertebrate visual phototransduction. The subcellular localization of the alpha subunit of visual Gq in crayfish photoreceptor was investigated immunocytochemically and biochemically to demonstrate the details of the rhodopsin-Gq interaction. The localization of Gq(alpha) changed depending on the light condition. In the dark, Gq(alpha) was localized in the whole rhabdoms as the membrane-bound form. In the light, half of the Gq(alpha) was localized in the cytoplasm as the soluble form. The translocation of Gq(alpha) was reversible. The light-modulated translocation possibly controls the amount of Gq that can be activated by rhodopsin. In vitro hydroxylamine treatment of rhabdomeric membranes suggested that the translocation was regulated by the fatty-acid modification of Gq(alpha).

Presence of retina-specific proteins in the lamprey pineal complex.

The pineal complex of river lamprey reacted with the antisera raised against retina specific proteins including bovine opsin, chick visinin and frog light-sensitive cyclic GMP phosphodiesterase (PDE). Immunoreactive materials stained with anti-opsin were evenly located at the outer segment of photoreceptor cells in the pineal organ and also found in the parapineal organ. Although anti-visinin stained the pineal and parapineal photoreceptor cells, the immunopositive photoreceptor cells were observed only at the lateral portion and not at the medial portion of the pineal organ. No immunoreactive materials were found in the pineal complex by the anti-PDE, whereas the anti-PDE reacted with photoreceptor cells of the retinal tissue. The data suggest that the pineal and parapineal retinas of lamprey contain opsin- and visinin-like proteins with different distribution in their photoreceptor cell layer as found in the lamprey retinal tissue.

Melatonin Receptors in the Spinal Cord

The pineal hormone, melatonin, plays an important role in the regulation of diurnal and seasonal rhythms in animals. In addition to the well established actions on the brain, the possibility of a direct melatonin action on the spinal cord has to be considered. In our laboratory, we have obtained data suggesting that melatonin receptors are present in the spinal cords of birds and mammals. Using radioreceptor binding and quantitative autoradiography assays with 2-[125I]iodomelatonin as the specific melatonin agonist, melatonin binding sites have been demonstrated in the rabbit and chicken spinal cords. These sites are saturable, reversible, specific, guanosine nucleotide-sensitive, of picomolar affinity and femtomolar density. The linearity of Scatchard plots of saturation data and the unity of Hill coefficients indicate that a single class of melatonin binding sites is present in the spinal cord membranes studied. The picomolar affinity of these sites is in line with the circulating levels of melatonin in these animals suggesting that these sites are physiologically relevant. Autoradiography studies in the rabbit spinal cord show that melatonin binding sites are localized in the central gray substance (lamina X). In the chicken spinal cord, these binding sites are localized in dorsal gray horns (laminae I-V) and lamina X. As lamina X and laminae I-II have similar functions, melatonin may have comparable roles in the chicken and rabbit spinal cords. Moreover, in the chicken spinal cord, the density of 2-[125I]iodomelatonin binding in the lumbar segment was significantly higher than those of the cervical and thoracic segments. The densities of these binding sites changed with environmental manipulations. When chickens were adapted to a 12L/12D photoperiod and sacrificed at mid-light and mid-dark, there was a significant diurnal variation in the density (maximum number of binding sites; Bmax) of melatonin binding sites in the spinal cord. After constant light treatment or pinealectomy, the Bmax of melatonin receptors in the chicken spinal cord increased significantly in the subjective mid-dark period. Moreover, there was an age-related decrease in the 2-[125I]iodomelatonin binding to the chicken spinal cord. Our results suggest that melatonin receptors in the chicken spinal cord are regulated by environmental lighting and change with development. These receptors may play an important role in the chronobiology of spinal cord function. The biological responses of melatonin on spinal cords have also been demonstrated in vitro. Melatonin decreased the forskolin-stimulated cAMP production in the chicken spinal cord explant. Preincubation with pertussis toxin blocked the melatonin effect. Our results suggest that melatonin receptors in the chicken spinal cord are linked to the adenylate cyclase via a pertussis toxin-sensitive G protein and that melatonin binding sites in spinal cords are melatonin receptors with biological functions. These receptors may be involved in the regulation of spinal cord functions related to sensory transmission, visceral reflexes and autonomic activities.

Identification and characterization of the cell-associated binding protein for urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI) inhibits not only tumor cell invasion but also production of experimental and spontaneous metastasis. Cell-binding experiments indicated that human choriocarcinoma SMT-cc1 cells have specific binding sites for UTI on their cell surface. [Kobayashi et al., J. Biol. Chem. 269, 1994, 20,642-20,647]. UTI binding protein (UTIBP) was purified to homogeneity by a combination of UTI-coupled affinity beads, preparative polyacrylamide gel electrophoresis and reverse phase HPLC. This protein is very similar to a truncated form of human cartilage link protein (LP). LP was identified structurally by its apparent molecular mass with and without deglycosylation treatment: Immunologically by the reactivity with anti-UTIBP antibody, and functionally by its ability to bind the NH2-terminal domain of UTI. UTI and UTIBP are distributed uniformly in the cytoplasm and/or over the cell surface of tumor cells and fibroblasts. The level of staining for hyaluronic acid, UTIBP and UTI is much lower in sections digested with hyaluronidase. These results suggest that the cell membrane-derived UTI-associated binding protein is the LP of proteoglycan-hyaluronic acid aggregates, which interacts with hyaluronic acid. Cell-associated LP may play a role in modulating protease activity to the environment close to tumor and fibroblast cell surface.

Photoreceptor cells and neural elements with long axonal processes in the pineal organ of the lamprey, Lampetra japonica, identified by use of the horseradish peroxidase method

SummaryHorseradish peroxidase (HRP) was applied to the transected end of the pineal tract of the lamprey, Lampetra japonica. Distinct reaction products of HRP were observed in 2 types of cell other than ganglion cells. The first type of cell protrudes a knob-like process into the pineal lumen. This type of cell was clearly identified by electron microscopy as a photoreceptor cell; its outer segment was connected to the ellipsoid through a sensory cilium. The other type of cell was located among photoreceptor and supporting cells. The processes of these cells were thin and slender, and they obviously did not represent photoreceptor, supporting, or conventional ganglion cells. The present results indicate that, in the lamprey, some of the photoreceptor cells of the pineal organ project their axon-like processes toward the posterior commissure, but that there is also another type of cell displaying long axonal projections. HRP-containing cells were distributed randomly over the pineal organ and were occasionally also observed in the parapineal organ.

Blue sensitive visual pigment and photoregeneration in pineal photoreceptors measured by high performance liquid chromatography

Abstract 1. 1. Chromophores of visual pigment in the lamprey pineal organ were examined using high performance liquid chromatography (HPLC). 2. 2. They consisted of mainly 3-dehydroretinal with a small amount of retinal; the ratio between the two chromophores was constant in different seasons. 3. 3. No significant change in chromatograms was observed between samples prepared in the dark and after irradiation by orange light (> 560 nm). 4. 4. However, some of the visual pigments were bleached by blue light (420 nm). 5. 5. Irradiation to orange light following cis-to-trans isomerization by blue light caused trans-to-cis isomerization. 6. 6. In the pineal photoreceptor these results suggest that blue sensitive visual pigments are contained, and that photoregeneration occurred.

Retinal isomers in the cerebral ganglion of the ascidian, Halocynthia roretzi

Summary The chromophore of photopigments was found in the ganglion of the solitary ascidian, Halocynthia roretzi, using the retinaloxime method with high performance liquid chromatography (HPLC) technique. Irradiation with orange light of minced ganglia causes photoisomerization of all-trans-retinaldehyde to 11-cis-retinaldehyde. Inversely, blue light irradiation of the sample which was irradiated with orange light isomerizes 11-cis-retinaldehyde to all-trans-retinaldehyde. We propose that the ganglion is a possible photoreceptor that is related to gamete release in the adult ascidian.

Three-Dimensional Reconstruction of Serotonin-Immunoreactive Photoreceptors in the Pineal Organ of the River Lamprey, Lampetra japonica

Serotonin-immunoreactive (5-HT IR) photoreceptors are present in the pineal complex (pineal and parapineal organ) of the river lamprey, Lampetra japonica. They are so-called modified pineal photoreceptors and have been regarded as photoneuroendocrine cells which secrete melatonin. We reconstructed 5-HT IR cells with a computer to demonstrate their three-dimensional structures from optical sections taken by a confocal laser scanning microscope. The 5-HT IR cell possesses a basal process, and it appears that the process does not branch out. These processes contact each other at the basal region of the end vesicle, and a process extends to the soma of the neighboring 5-HT IR cell. These findings were obtained by three-dimensional analysis with a computer, which is a useful technique to demonstrate the interaction between cells. We suggest that the 5-HT IR photoreceptors interact with one another.

Melatonin Excretion Rhythms in the Cultured Pineal Organ of the Lamprey, Lampetra japonica

Pineal organ of the lamprey, Lampetra japonica, is essential to keep the circadian locomotor activity rhythm as previously reported. In this paper, we tried to show that an endogenous oscillator is located and is working in the pineal organ. When the pineal organs were excised and cultured in a plastic tube with M199 medium at 20 degrees C, melatonin secretion rhythms were clearly observed under both light-dark and continuous dark conditions. The circadian secretion of melatonin continued for more than five cycles under the continuous dark condition. This indicates that the pineal organ has an endogenous oscillator and that the melatonin secretion rhythm is controlled by this oscillator. These findings suggest the possibility that the locomotor activity rhythm of the lamprey is under the control of the oscillator in the pineal organ.