Kazunaga Takazawa

Inositol 1, 4, 5-trisphosphate 3-kinase distribution in the rat brain. High levels in the hippocampal CA1 pyramidal and cerebellar Purkinje cells suggest its involvement in some memory processes

The distribution of inositol 1,4,5-trisphosphate (InsP3) 3-kinase was studied in the adult rat brain, using polyclonal antibodies raised against the purified 50,000-Da rat brain enzyme by immunohistochemistry and Western blot, in addition to enzymatic assay. Immunohistochemically, the enzyme was detected in neurons, where it was localized in the dendrites and at the periphery of the cell bodies. Using selective toxin lesions, the highest enzyme levels were found in the dendrites of hippocampal CA1 pyramidal cells and in neurons in the dorsal portion of the lateral septum, regions both involved in long-term potentiation; and in the dendrites of Purkinje cell subpopulations in the cerebellum, a region involved in long-term depression. High levels were found in neurons in the cortex; in the anterior olfactory nucleus; in the striatum (caudate, putamen, olfactory tubercle, Calleja islets and accumbens); in the central nucleus of the amygdala; in the hippocampal dentate gyrus and in the subiculum. The enzyme was not detected in other brain regions. By Western blot, a 50,000-Da immunoreactive band was present in the cortex, caudate-putamen and cerebellum. This band was most highly stained in the hippocampus. InsP3 3-kinase activity, stimulated by calcium/calmodulin, corresponded to 6172-2638 pmol of InsP4 produced/min/mg protein in the hippocampus followed by frontal and parietotemporal cortex and cerebellum. This activity was below 400 in the brainstem and spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and expression of a human brain inositol 1,4,5-trisphosphate 3-kinase

A human hippocampus cDNA library was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA. Sequencing of three overlapping clones identified a 1383 bp open reading frame encoding a 461 amino acid protein with a calculated molecular weight of 50988. The coding amino acid sequence showed an overall 93% similarity with the sequence of rat brain InsP3 3-kinase. The cDNA insert of one isolated partial clone (i.e. hh 26) was in frame with the beta galactosidase fragment fused to it as a Bluescript plasmid; it displayed InsP3 3-kinase activity when expressed in Escherichia coli (E. coli). Biochemical characterization of human brain InsP3 3-kinase by SDS polyacrylamide gel electrophoresis and regeneration of enzyme activity reveals three active fractions with apparent Mr of 58,000-64,000, 45,000-50,000 and 37,000-39,000.

Immunochemical and Immunohistochemical Localization of Parvalbumin in Rat Nervous Tissues

Abstract: The contents of parvalbumin in various nervous tissues of the rat were measured by radioimmunoassay (RIA) and its cellular distribution was immuno‐histochemically examined by peroxidase‐antiperoxidase methods. The antibody, raised in rabbits using rat skeletal muscle parvalbumin, did not cross‐react with other Ca2+ ‐binding proteins such as calmodulin or S‐100 proteins. The RIA demonstrated the wide distribution of the antigen, with very high levels in the cerebellum (3.217 ± 519 ng/mg protein). The immunohistochemical description by Celio and Heizmann [Nature 293, 300‐302 (1981)] was confirmed concerning the existence of the antigen in Purkinje cells of the cerebellum; nonpyramidal neurons of the cerebral cortex; and medium‐sized cells of the olfactory bulb, hippocampus, and reticular nucleus of the thalamus. In addition to these neurons, we found the parvalbumin‐like immunoreactivity in the large neurons of the superior vestibular nucleus and the neurons of the medial superior olive nucleus. In the γ‐aminobutyric acid (GABA)‐containing nuclei such as substantia nigra, caudatoputamen, and globus pallidus. parvalbumin‐positive cells and fibers were rare. In the medial lemniscus of the midbrain which contains no GABA, parvalbumin‐immunoreactive fibers were prominent. The possibility was discussed that parvalbumin exists in a specific population of neurons that differ from those containing GABA.

Inositol 1,4,5-Trisphosphate 3-Kinase mRNA: High Levels in the Rat Hippocampal CA1 Pyramidal and Dentate Gyrus Granule Cells and in Cerebellar Purkinje Cells

Abstract: The distribution of inositol 1,4,5‐trisphosphate (InsP3) 3‐kinase mRNA in the rat brain is reported using oligonucleotides based on a cDNA clone sequence that encodes rat brain InsP3 3‐kinase and the in situ hybridization technique. Moderate levels were found in CA2‐4 pyramidal neurons, in the cortex, and in the striatum. The cerebellar granule cells, thalamus, hypothalamus, brainstem, spinal cord, and white matter tracts were almost negative. The levels of InsP3 3‐kinase mRNA were highest in the hippocampal CA1 pyramidal neurons, granule cells of the dentate gyrus, and cerebellar Purkinje cells. These results contrast with the lower concentration of the InsP3 receptor already reported in the hippocampus versus the Purkinje cells and suggest a special role for inositol 1,3,4,5‐tetrakisphosphate in Ammons horn.

Ca2+/Calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase in rat and bovine brain tissues

Inositol 1,4,5-trisphosphate (Ins P3) 3-kinase catalyzes the ATP-dependent phosphorylation of Ins P3 to Inositol 1,3,4,5-tetrakisphosphate (Ins P4). Ca2+/calmodulin (CaM)-sensitivity of Ins P3 3-kinase was measured in the crude soluble fraction from rat brain and different anatomic regions of bovine brain. Kinase activity was inhibited in the presence of EGTA (free Ca2+ below 1 nM) as compared to Ca2+ (10 microM free Ca2+) or Ca2+ (10 microM free Ca2+) and CaM (1 microM). Ca2+-sensitivity was also seen for the cAMP phosphodiesterase measured under the same assay conditions, but was not for the Ins P3 5-phosphatase. DEAE-cellulose chromatography of the soluble fraction of rat brain or bovine cerebellum resolved a Ca2+/CaM-sensitive Ins P3 3-kinase (maximal stimulation at 1 microM Ins P3 substrate level was 2.0-3.0 fold).

Inositol 1,4,5-trisphosphate 3-kinase highest levels in the dendritic spines of cerebellar Purkinje cells and hippocampal CA1 pyramidal cells. A pre- and post-embedding immunoelectron microscopic study.

Inositol 1,4,5-trisphosphate 3-kinase (InsP3 3-kinase) plays a crucial role in calcium homeostasis by regulating InsP3 levels. We have reported the highest concentrations of InsP3 3-kinase in the dendrites of cerebellar Purkinje cells and hippocampal pyramidal cells of the CA1 sector of the Ammons horn. We here investigate its subcellular localization by pre- and post-embedding immunoelectron microscopic study. In both populations of neurons, the major structure expressing a high level of InsP3 3-kinase is the dendritic spines.

Comparison of neuronal inositol 1,4,5-trisphosphate 3-kinase and receptor mRNA distributions in the human brain using in situ hybridization histochemistry

The distribution of the messenger RNA coding for the recently cloned inositol 1,4,5-trisphosphate (InsP3) 3-kinase, the enzyme phosphorylating InsP3 to InsP4, was compared to the localizations of InsP3 receptor mRNA in the human brain using in situ hybridization histochemistry and oligonucleotide probes. InsP3 3-kinase and receptor mRNA levels were high in the cerebellar Purkinje cells. They were also observed, to a much lesser degree than in the cerebellum, in the hippocampal CA1 pyramidal cells and dentate gyrus granule cells, in the majority of the cortical neurons and in the striatal medium-sized neurons. Both mRNAs were not detected in the brainstem and in the glial cells.

Comparison of neuronal inositol 1,4,5-trisphosphate 3-kinase and receptor mRNA distributions in the adult rat brain using in situ hybridization histochemistry.

As a result of its interaction with a specific receptor, inositol 1,4,5-trisphosphate mobilizes intracellular calcium. The metabolism of inositol 1,4,5-trisphosphate is rather complex: inositol 1,4,5-trisphosphate 3-kinase produces inositol 1,3,4,5-tetrakisphosphate, a putative second messenger. In order to elucidate inositol 1,3,4,5-tetrakisphosphate function, a comparative in situ hybridization study of the distributions of inositol 1,4,5-trisphosphate 3-kinase and receptor mRNAs was performed in the adult rat brain using oligonucleotides derived from their cDNA sequences. The neuronal distributions of the mRNA for the receptor were larger than for the kinase. Highest levels of both mRNAs were found in the cerebellar Purkinje cells, where they were enriched in their neuronal perikarya and to a lesser extent in their dendrites. In addition to the cerebellum, mRNAs were mainly detected in the hippocampal pyramidal cells of the CA1 sector of the Ammons horn and in the granule cells of the dentate gyrus, and also in a majority of the neurons in the cortical layers II-III and V, especially in the frontal cortex and cingulate cortex; caudate-putamen, accumbens, olfactory tubercle and Calleja islets; claustrum; anterior olfactory nucleus; taenia tecta; piriform cortex; dorsolateral septum; bed nucleus stria terminalis; amygdala; hippocampal CA2-4 sectors and subiculum. The inositol 1,4,5-trisphosphate receptor mRNA but not kinase mRNA was found in a majority of the neurons in the thalamus, especially in the parafascicular nucleus; hypothalamus, especially the medial hypothalamus; substantia nigra pars compacta and ventral tegmental area; superior colliculus; lateral interpeduncular nucleus and central gray. Taking into account the limitation in sensitivity of the technique, both mRNAs were not detected in glial cells and in the olfactory bulb; basal nucleus of Meynert, diagonal band nuclei; medial septal nucleus; substantia innominata; globus pallidus; entopeduncular nucleus; substantia nigra pars reticulata; ventral pallidum; subthalamic nucleus; spinal cord and dorsal root ganglia. In conclusion, cerebellum and hippocampus appear to contain almost similar levels of kinase mRNA. This is in contrast to receptor mRNA levels which were at much higher levels in the cerebellum when compared with the hippocampus. For this reason, we have chosen hippocampal CA1 pyramidal cells and dentate gyrus granule cells for studying inositol 1,4,5-trisphosphate 3-kinase function.

T4-thyroid storm after CT-scan with iodinated contrast medium

A 62-year-old woman had thyroid storm 5 h after a CT examination (contrast enhancement with 65% meglumine amidotrizoate, Angiografin). At the time of admission to our hospital, serum T4 and serum free T4 levels were markedly elevated (29 μg/dl and 9.6 ng/dl, respectively); serum T3 and serum free T3 levels were within normal limits (144 ng/dl and 5.9 pg/ml, respectively). These findings suggest that thyroid storm can occur with excess T4 only (T4-thyroid storm), and that T4-thyroid storm can be caused by administration of iodinated contrast medium.

Identification of high molecular weight forms of inositol 1,4,5-trisphosphate 3-kinase in rat thymus and human lymphocytes

A soluble inositol 1,4,5-trisphosphate 3-kinase (InsP3 3-kinase) has been characterized from extracts of rat thymus. The enzyme was shown to have a molecular weight within the range 98,000-114,000 M(r) as determined by regeneration of enzyme activity from sodium dodecyl sulphate polyacrylamide gels. The enzyme phosphorylates inositol 1,4,5-trisphosphate (InsP3) to inositol 1,3,4,5-tetrakisphosphate (InsP4) with an apparent Km of 3.1 +/- 0.4 microM. The enzyme is stimulated 4-6-fold by Ca2+/calmodulin and is not recognised by polyclonal antisera raised against rat brain InsP3 3-kinase A. High levels of InsP3 3-kinase activity were also detected in soluble extracts of human lymphocyte preparations. The human lymphocyte enzyme was shown to have a molecular weight between 61,000 and 70,000 M(r) as judged by SDS-PAGE, and was stimulated approximately 10-fold in the presence of Ca2+/calmodulin. These results establish that InsP3 3-kinase from rat thymus and human lymphocyte preparations represent high molecular weight isoenzymes of the InsP3 3-kinase family.