Yasuhiko Matsukado

Dephosphorylation of microtubule-associated protein 2, τ factor, and tubulin by calcineurin

Abstract: Calcineurin dephosphorylated microtubule‐associated protein 2 (MAP2) and τ factor phosphorylated by cyclic AMP‐dependent and Ca2+, calmodulin‐dependent protein kinases from the brain. Tubulin, only phosphorylated by the Ca2+, calmodulin‐dependent protein kinase, served as substrate for calcineurin. The concentrations of calmodulin required to give half‐maximal activation of calcineurin were 21 and 16 nM with MAP2 and τ factor as substrates, respectively. The Km and Vmaxvalues were in ranges of 1–3 μM and 0.4–1.7 μmol/mg/ min, respectively, for MAP2 and τ factor. The Km value for tubulin was in a similar range, but the Vmax value was lower. The peptide map analysis revealed that calcineurin dephosphorylated MAP2 and τ factor universally, but not in a site‐specific manner. The autophosphorylated Ca2+, calmodulin‐dependent protein kinase was not dephosphorylated by calcineurin. These results suggest that calcineurin plays an important role in the functions of microtubules via dephosphorylation.

The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay.

Calcineurin is the calcium (divalent cations)-dependent calmodulin-stimulated phosphoprotein phosphatase which is capable of dephosphorylating various substrate proteins. The subcellular and regional distribution of calcineurin in the rat brain has been studied by light and electron microscopic immunohistochemistry using antiserum against calcineurin. Immunoreactivity was observed in many neurons but was not detected in glial cells, such as astrocytes, oligodendrocytes and ependymal cells by the PAP method. Light microscopy demonstrates strong immunoreactivity in neuronal somata and neurites. By electron microscopy, calcineurin immunoreactivity was found to be present in dendrites including postsynaptic densities, somata, spines, axons and terminals. Calcineurin immunoreactivity was present in neurons throughout the brain, but a marked regional variation in strength of the immunoreactivity was observed. The caudatoputamen, hippocampal formation, and substantia nigra were strongly stained. Cerebral and cerebellar neocortex showed moderate immunoreactivity. In substantia nigra and globus pallidus, only neurites were stained, but neuronal somata not. The staining of the substantia nigra was thought to be due to that of the nerve terminals originating from the caudatoputamen, in view of the findings by cerebral hemitransection and electron microscopic immunohistochemistry. We developed an enzyme-immunoassay (EIA) for calcineurin. The sensitivity of the EIA was 1 ng (13 fmol) of calcineurin. We determined the level of calcineurin in various regions of the rat brain. The caudate nucleus, putamen and hippocampal formation showed a high concentration of calcineurin. The results are consistent with those obtained by immunohistochemistry.

Morphological characterization of the rat striatal neurons expressing calcineurin immunoreactivity.

Calcineurin, a multifunctional Ca2+ (divalent cations)-dependent calmodulin-stimulated phosphoprotein phosphatase, has been reported to be present in the striatal neurons which project to the globus pallidus and the substantia nigra. In the present study, we examined what types of cells in the rat striatum express calcineurin. The calcineurin-positive neurons were of medium size (mean diameter of 16 microns) and constituted about 60-70% of the total neuronal population in the striatum. Under light microscopy, the calcineurin-positive neurons had round, triangular, or polygonal cell bodies with a relatively small amount of cytoplasm. Electron microscopic examination of 20 randomly selected striatal calcineurin-immunoreactive neurons revealed that their nuclei did not show any invaginations or intranuclear inclusions. The calcineurin-positive neurons were characterized by Golgi impregnation as the densely spinous type. On the other hand, it was demonstrated that calcineurin-positive neurons are a separate population from the diisopropylfluorophosphate-acetylcholinesterase-positive cells or nicotinamide adenine dinucleotide phosphate diaphorase-positive cells, by means of the combination of immunocytochemistry and enzyme histochemistry. In addition, simultaneous localization of calcineurin and substance P in a single cell was observed in some striatal neurons using a double immunostaining method. On the basis of these findings, it was considered that most calcineurin-immunoreactive neurons in the rat striatum may be classified as medium-size densely spiny neurons.

Intracerebral venous angioma with arterial blood supply: A mixed angioma

An 18-year-old man was operated upon with a diagnosis of intracerebral hematoma. Although the angiogram of the right carotid artery demonstrated the characteristic umbrella appearance of the venous angioma in the parietal lobe, fine arterial branches supplying the vascular malformation were noted in the preoperative angiogram. At the operation, no nidus was found, and delicate angiomatous networks with red veins were observed around the hematoma cavity. Histologic examination of the angiomatous network proved it to be an arteriovenous malformation. It was a mixed angioma, which combined venous angioma and arteriovenous malformations. Clinical, angiographic, and pathological features of intracerebral venous angiomas with arterial components were reviewed from the literature.

Inactivation and Reactivation of the Multifunctional Calmodulin-Dependent Protein Kinase from Brain by Autophosphorylation and Dephosphorylation: Involvement of Protein Phosphatases from Brain

Abstract: The multifunctional calmodulin‐dependent protein kinase (calmodulin‐kinase) from rat brain was autophosphorylated in a Ca2+‐and calmodulin‐dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin‐kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin‐kinase were universally dephosphorylated by protein phosphatase C. Calmodulin‐kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin‐kinase by autophosphorylation and dephosphorylation was observed using calmodulin‐kinase from canine heart. These results suggest that the activity of calmodulin‐kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin‐kinases in various tissues.

The response of human cerebrum adenyl cyclase to biogenic amines.

THE PRIMARY site of action of various hormones has been shown to be the enzyme adenyl cyclase, which catalyses the formation of adenosine 3’,5’-monophosphate (cyclic 3’,5’-AMP) from ATP (S-RLAND, OYE and BUTCHER, 1965). The occurrence of this enzyme is ubiquitous and the highest activity among organs investigated is reported for brain ( S ~ R L A N D , RALL and MENON, 1962). The cyclase appears to possess an effector site which responds to a hormone. The effect of a hormone is limited to the adenyl cyclase present in the target organ for the hormone (ROBMSON, BUTCHER and SUTHEIUAND, 1967). Studies with laboratory animals have shown that the brain adenyl cyclase responds to various biogenic amines, such as norepinephrine, histamine, and serotonin (KAKIUCHI and RALL, 1968~. b; S ~ J Z U , DALY and CREWLING 1969). In cerebrum of rabbit and guinea pig, histamine is a more potent activator of the adenyl cyclase than norepinephrine, whereas the reverse situation is reported for rat cerebrum and rabbit cerebellum. It has been widely assumed that some biogenic mines function as neurohormones or chemical transmitters in human brain as well as in the brain of other animals. This assumption is mainly based on two findings: (1) the occurrence of these m i n e s and related enzymes in human brain; and (2) changes in human behaviour and/or emotion after administration of a drug which is known to affect concentrations of the amines in animal brains. Little is yet known about the biochemical mechanism of action of the endogenous amines in animal or human brain. An interesting speculation on the mechanism is that the central effect of some biogenic amines may be, at least in part, mediated by cyclic 3‘,5’-AMP. Accumulating data with laboratory animals now substantiate this hypothesis (GOLDBERG and SINGER, 1969; MCKEAN, PETERSON and RAUGHPATHY, 1969; SIGGMS, HOFPW and BLOOM, 1969; SHIMIZU et a!., 19706). The hypothesis could be extended to human brain if any effect of the amines on the cyclic 3’,5’-AMP system in the human brain were demonstrated. We investigated this possibility with intact tissue of human cerebrum.

The cyclic AMP system of human brain

In the cortical gray matter of human brains, which were obtained by permissible resection at the time of surgery, cyclic 3′, 5′-AMP was found in a range of concentrations between 42 and 143 pmole/mg protein. The subcortical white matter and various types of brain tumor contained much lower concentrations of the nucleotide. The level of the cyclic nucleotide in incubated slices of gray matter was elevated 20–50-fold by norepinephrine (0.5–1.0 mM) or veratridine (0.05 mM), 4–7-fold by histamine (1.0mM) or adenosine (0.2mM) and 1.5–2-fold by serotonin. With respect to the stimulatory effect on the cyclic AMP level in human brain slices, norepinephrine was inferior to epinephrine or isoproterenol, and was completely antagonized with a β-adrenergic blocking agent, propranolol, but only slightly by α-blocking agents, such as phentolamine and dibenamine. The particulate fraction of human brain homogenate was capable of synthesizing cyclic AMP from ATP. This enzymic activity was 0.19–0.24 nmole/min/mg protein. This activity was stimulated 2–3-fold by the addition of 10mM NaF but not by any of the biogenic amines tested. The activity of nucleotide phosphodiesterase of human brain was almost equal to that of rat brain: only a single Km for the substrate cyclic AMP was observed around 0.9 × 10−4M.

Occlusion of the internal carotid artery after radiation therapy for the chiasmal lesion

SummaryOcclusion of the intracranial portion of the internal carotid artery were encountered in two cases with suprasellar tumours, who had received irradiation therapy previously and shown excellent clinical improvement postoperatively.Clinical features and the angiographic characteristics were analysed in these two cases and the restults were compared with reports in the literature. The occlusive effect of radiation on the arterial wall was conceived as the cause of carotid obstruction. In order to avoid this side effect of radiation therapy, especially for brain tumours of low malignancy in childhood, careful estimation of the radiation dose should be required and cerebral angiographic follow-up during and after the treatment with radiation is indicated, particularly when the patient develops cerebral transient ischaemic symptoms.

Stimulated formation of adenosine 3′,5′-monophosphate by desipramine in brain slices

Abstract The effect of five tricyclic psychotropic drugs on the formation of adenosine 3′,5′-monophosphate (cyclic AMP) was examined using guinea pig cerebral slices which had been preincubated with [8-14C]adenine to label a proof of intracellular ATP. Three antidepressants, i.e. imipramine, desipramine, and amitriptyline elevated the level of cyclic [14C]AMP and increased the relative formation of cyclic [14C]AMP in the slices more than 5-fold. Two tranquilizers, chlorpromazine and perfenazine had little effect. The effect of desipramine was maximal at 0.5 mM, was blocked by 1.0 mM theophylline by more than 80%, and was not inhibited by either phentolamine or propranolol.

Localization of Glycogen Synthase in Brain

Antisera against glycogen synthase from canine brain were prepared and used for investigation of the localization of the enzyme in the brain. Antisera cross‐reacted only with the 88‐kilodalton protein that is the subunit of brain glycogen synthase. Immunoreactivity of glycogen synthase was universally distributed in all regions of the brain, although hippocampus, cerebral cortex, caudatoputamen, and cerebellar cortex had relatively high immunoreactivity. Light microscopic examination revealed that the immunoreactivity was found in all cell types, such as neurons in several regions, astrocytes, ependymal cells surrounding the ventricle, oligodendrocytes, and epithelial cells of the choroid plexus in the ventricle. Immunoreactive intensity was more prominent in neurons than glial cells. Immunostaining may be a useful tool for investigation of the state of glycogen metabolism under normal and pathological conditions.