Hitoshi Michibata

THE ACCUMULATION AND DISTRIBUTION OF VANADIUM, IRON, AND MANGANESE IN SOME SOLITARY ASCIDIANS

The vanadium, iron, and manganese contents of 15 species of solitary ascidians belonging to the suborders Phlebobranchia and Stolidobranchia were determined by thermal neutron activation analysis. Vanadium was detectable in all species examined. In general, the vanadium content in various tissues of the Phlebobranchia was considerably higher than the iron and manganese contents. The blood cells especially contained a large amount of vanadium. The highest value (21 µg vanadium/mg dry weight) was obtained from blood corpuscles of Ascidia ahodori. Species in the suborder Stolidobranchia, on the other hand, had smaller quantities of vanadium in comparison with those in the suborder Phlebobranchia. The iron and manganese contents did not differ greatly between the two suborders. The data are considered in the light of physiological roles of these transition metals in ascidians.

Molecular biological approaches to the accumulation and reduction of vanadium by ascidians

Abstract About 90 years ago, Henze discovered high levels of vanadium in the blood (coelomic) cells of an ascidian collected from the Bay of Naples. His discovery attracted the interdisciplinary attention of chemists, physiologists, and biochemists. Two decades ago, we quantified the vanadium levels in several ascidian tissues definitively using neutron-activation analysis and revealed that some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, corresponding to about 107 times that found in seawater. Vanadium accumulated is reduced to the +3 oxidation state via the +4 oxidation state and stored in vacuoles of vanadocytes (vanadium-containing blood cells) where high levels of protons and sulfate are also contained. To investigate this unusual phenomenon, we isolated several proteins and genes that are expressed in vanadocytes. To date, three types of vanadium-binding protein, designated as Vanabins, have been isolated, with molecular masses of 12.5, 15, and 16 kDa, along with the cDNAs encoding these proteins. In addition, four types of enzyme related to the pentose phosphate pathway that produces NADPH were revealed to be located in vanadocytes. The pentose phosphate pathway participates in the reduction of V(V) to V(IV). The cDNA for each of the vacuolar-type H+ATPase (VATPase) A, B, C, and D subunits, which are located on the vacuolar membranes of vanadocytes, has been isolated and analyzed. VATPase generates a proton-motive force, and is thought to provide the energy for vanadium accumulation. To clarify the entire mechanism involved in the accumulation and reduction, much more genes and proteins expressed in the blood cells need to be systematically identified. Thus, we have performed an expressed sequence tag (EST) analysis of blood cells and have established the functional assay system to elucidate the functions of genes and proteins obtained from ascidian blood cells.

Vanadium-binding proteins (Vanabins) from a vanadium-rich ascidian Ascidia sydneiensis samea

Since the beginning of the last century, it has been known that ascidians accumulate high levels of a transition metal, vanadium, in their blood cells, although the mechanism for this curious biological function remains unknown. Recently, we identified three vanadium-binding proteins (vanabins), previously denoted as vanadium-associated proteins (VAPs) [Zool. Sci. 14 (1997) 37], from the cytoplasm fraction of vanadium-containing blood cells (vanadocytes) of the vanadium-rich ascidian Ascidia sydneiensis samea. Here, we describe the cloning, expression, and analysis of the metal-binding ability of vanabins. Recombinant proteins of two independent but related vanabins, vanabin1 and vanabin2, bound to 10 and 20 vanadium(IV) ions with dissociation constants of 2.1x10(-5) and 2.3x10(-5) M, respectively. The binding of vanadium(IV) to these vanabins was inhibited by the addition of copper(II) ions, but not by magnesium(II) or molybdate(VI) ions. Vanabins are the first proteins reported to show specific binding to vanadium ions; this should provide a clue to resolving the problem regarding the selective accumulation of vanadium in ascidians.

l‐Aspartate but Not the d Form Is Secreted Through Microvesicle‐Mediated Exocytosis and Is Sequestered Through Na+‐Dependent Transporter in Rat Pinealocytes

Abstract: Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine‐stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca2+ ionophore) in the presence of Ca2+ and was proportional to the amount of l‐glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L‐ or N‐type voltage‐gated Ca2+ channel blockers. Bay K 8644, an agonist for the L‐type Ca2+ channel, stimulated the release of aspartate 2.1‐fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the l form, although pinealocytes also contain the d form in their cytoplasm, corresponding to ∼30% of the total free aspartate. Radiolabeled l‐aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na+‐dependent manner. The Na+‐dependent uptake of l‐aspartate was strongly inhibited by l‐cysteine sulfinate, β‐hydroxyaspartate, and l‐serine‐O‐sulfate, inhibitors for the Na+‐dependent glutamate/aspartate transporter on the plasma membrane. Na+‐dependent sequestration of l‐aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that l‐aspartate is released through microvesicle‐mediated exocytosis from pinealocytes and is taken up again through the Na+‐dependent transporter at the plasma membrane. The possible role of l‐aspartate as an intercellular chemical transmitter in the pineal gland is discussed.

Microvesicle-mediated exocytosis of glutamate is a novel paracrine-like chemical transduction mechanism and inhibits melatonin secretion in rat pinealocytes.

Abstract: Mammalian pinealocytes are neuroendocrine cells that synthesize and secrete melatonin, these processes being positively controlled by norepinephrine derived from innervating sympathetic neurons. Previously, we showed that pinealocytes contain a large number of microvesicles (MVs) that specifically accumulate L‐glutamate through a vesicular glutamate transporter and contain proteins for exocytosis such as synaptobrevin 2 (VAMP2). These findings suggested that the MVs are counterparts of synaptic vesicles and are involved in paracrine‐like chemical transduction in the pineal gland. Here, we show that pinealocytes actually secrete glutamate upon stimulation by KC1 in the presence of Ca2+ at 37°C. The ability of glutamate secretion disappeared when the cells were incubated at below 20°C. Loss of the activity was also observed on successive stimulation, but it was recovered after 12 hr incubation. A low concentration of cadmium chloride or ω‐conotoxin GVIA inhibited the secretion. Botulinum neurotoxin E cleaved synaptic vesicle‐associated protein 25 (SNAP‐25) and thus inhibited the secretion. The released L‐glutamate stimulated pinealocytes themselves via glutamate receptor(s) and inhibited norepinephrine‐stimulated melatonin secretion. These results strongly suggest that pinealocytes are glutaminergic paraneurons, and that the glutaminergic system regulates negatively the synthesis and secretion of melatonin. The MV‐mediated paracrine‐like chemical transduction seems to be a novel mechanism that regulates hormonal secretion by neuroendocrine cells.

Scanning X-ray Microscopy of Living and Freeze-Dried Blood Cells in Two Vanadium-Rich Ascidian Species, Phallusia mammillata and Ascidia sydneiensis samea

Abstract Some ascidians (sea squirts) accumulate the transitional metal vanadium in their blood cells at concentrations of up to 350 mM, about 107 times its concentration found in seawater. There are approximately 10 different types of blood cell in ascidians. The identity of the true vanadium-containing blood cell (vanadocyte) is controversial and little is known about the subcellular distribution of vanadium. A scanning x-ray microscope installed at the ID21 beamline of the European Synchrotron Radiation Facility to visualize vanadium in ascidian blood cells. Without fixation, freezing or staining realized the visualization of vanadium localized in living signet ring cells and vacuolated amoebocytes of two vanadium-rich ascidian species, Phallusia mammillata and Ascidia sydneiensis samea. A combination of transmission and fluorescence images of signet ring cells suggested that in both species the vacuoles contain vanadium.

6-Phosphogluconate Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Form a Supramolecular Complex in Human Neutrophils That Undergoes Retrograde Trafficking during Pregnancy

Neutrophils from pregnant women display reduced neutrophil-mediated effector functions, such as reactive oxygen metabolite (ROM) release. Because the NADPH oxidase and NO synthase produce ROMs and NO, the availability of their substrate NADPH is a potential regulatory factor. NADPH is produced by glucose-6-phosphate dehydrogenase (G-6-PDase) and 6-phosphogluconate dehydrogenase (6-PGDase), which are the first two steps of the hexose monophosphate shunt (HMS). Using immunofluorescence microscopy, we show that 6-PGDase, like G-6-PDase, undergoes retrograde transport to the microtubule-organizing centers in neutrophils from pregnant women. In contrast, 6-PGDase is found in an anterograde distribution in cells from nonpregnant women. However, lactate dehydrogenase distribution is unaffected by pregnancy. Cytochemical studies demonstrated that the distribution of 6-PGDase enzymatic activity is coincident with 6-PGDase Ag. The accumulation of 6-PGDase at the microtubule-organizing centers could be blocked by colchicine, suggesting that microtubules are important in this enzyme’s intracellular distribution. In situ kinetic studies reveal that the rates of 6-gluconate turnover are indistinguishable in samples from nonpregnant and pregnant women, suggesting that the enzyme is functionally intact. Resonance energy transfer experiments showed that 6-PGDase and G-6-PDase are in close physical proximity within cells, suggesting the presence of supramolecular enzyme complexes. We suggest that the retrograde trafficking of HMS enzyme complexes during pregnancy influences the dynamics of NADPH production by separating HMS enzymes from glucose-6-phosphate generation at the plasma membrane and, in parallel, reducing ROM and NO production in comparison with fully activated neutrophils from nonpregnant women.

Direct reduction from vanadium(V) to vanadium(IV) by NADPH in the presence of EDTA. A consideration of the reduction and accumulation of vanadium in the ascidian blood cells

Abstract Vanadium(V) species are reduced to vanadium(IV) directly by NADPH in the presence of EDTA. At neutral pH, vanadium(V)–EDTA complex, [VO 2 (EDTA)] 3− (20 mM) was almost completely reduced to [VO(EDTA)] 2− directly by NADPH (200 mM) after 15 h under aerobic and anaerobic conditions. The reduction is markedly accelerated at low pH. At pH 3, the vanadium(V) complex (20 mM) was reduced by NADPH (20 mM) within 1 h. Oxygen had little effect, but inhibited the reduction to some extent at low pH. At low pH, simple vanadium(V) species were also partly reduced without the assistance of EDTA, resulting in the formation of mixed-valence (V(IV)–V(V)) polynuclear species. The accumulation and reduction of vanadium in the vanadocytes of ascidians are discussed based on the present results.

The L-type Ca2+ channel is involved in microvesicle-mediated glutamate exocytosis from rat pinealocytes

Abstract: Pinealocytes, parenchymal cells of the pineal gland, secrete glutamate through microvesicle‐mediated exocytosis upon depolarization by KC1 in the presence of Ca2+, which is involved in a novel paracrine‐like intercellular signal transduction mechanism in neuroendocrine organs. In the present study, we investigated whether or not the L‐type Ca2+ channel is involved in the microvesicle‐mediated glutamate secretion from cultured rat pinealocytes. Nifedipine, a specific antagonist of the L‐type Ca2+ channel, inhibited the Ca2+‐dependent glutamate exocytosis by 48% at 20 uM. Other L‐type Ca2+ channel antagonists, such as nitrendipine, showed similar effects. 1,4‐Dihydro‐2,6‐dimethyl‐5‐nitro‐4[2‐(trifluoromethyl)‐phenyl]‐3‐pyridinecarboxylic acid methyl ester (BAY K8644), an agonist of the L‐type Ca2+ channel, at 1 uM, on the other hand, stimulated the glutamate exocytosis about 1.6‐fold. Consistently, these Ca2+ channel antagonists inhibited about 50% of the Ca2+ uptake, whereas BAY K8644 increased the uptake 5.3‐fold. An antibody against the carboxyl‐terminal region of the rabbit L‐type Ca2+ channel recognized polypeptides of pinealocytes with apparent molecular masses of 250 and 270 kDa, respectively, and immunostained the plasma membrane region of the pinealocytes. These results strongly suggested that the entry of Ca2+ through L‐type Ca2+ channel(s), at least in part, triggers microvesicle‐mediated glutamate exocytosis in pinealocytes.

Vanadium in Ascidians

About 80 years ago Henze (1911) found unexpectedly that ascidian blood cells contained a high level of vanadium. The initial interest in this phenomenon was not only the presence of vanadium but also the possibility that other metals might be concentrated in ascidians and other organisms. Many analytical chemists subsequently analyzed the vanadium content of various organisms (Cantacuzene and Tchekirian, 1932; Vinogradov, 1934; Kobayashi, 1935; Webb, 1939; Noddack and Noddack, 1939; Bertrand, 1950; Lybing, 1953; Boeri and Ehrenberg, 1954; Webb, 1956; Levine, 1961; Bielig et al., 1961; Kalk, 1963a; 1963b; 1963c; Ciereszko et al., 1963; Bielig et al., 1966; Rummel et al., 1966; Carlisle, 1968; Swinehart et al., 1974; Danskin, 1978; Botte and Scippa, 1979; Michibata, 1984). Consequently, it became clear that the ascidians are the only organisms in animal kingdom known to accumulate high levels of vanadium and that all species among ascidians do not always contain large amounts of the metal.