Kazutoshi Kiuchi

The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells

We reported that one of the isoquinolinesulfonamide derivatives, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M. and Hidaka, H. (1990) J. Biol. Chem. 265, 4315-4320). We have now investigated the inhibitory property of a newly synthesized methoxybenzenesulfonamide, KN-93, on CaMKII activity in situ and in vitro. KN-93 elicited potent inhibitory effects on CaMKII phosphorylating activity with an inhibition constant of 0.37 microM but this compound had no significant effects on the catalytic activity of cAMP-dependent protein kinase, Ca2+/phospholipid dependent protein kinase, myosin light chain kinase and Ca(2+)-phosphodiesterase. KN-93 also inhibited the autophosphorylation of both the alpha- and beta-subunits of CaMKII. Kinetic analysis indicated that KN-93 inhibits CaMKII, in a competitive fashion against calmodulin. To evaluate the regulatory role of CaMKII on catecholamine metabolism, we examined the effect of KN-93 on dopamine (DA) levels in PC12h cells. The DA levels decreased in the presence of KN-93. Further, the tyrosine hydroxylase (TH) phosphorylation induced by KCl or acetylcholine was significantly suppressed by KN-93 in PC12h cells while events induced by forskolin or 8-Br-cAMP were not affected. These results suggest that KN-93 inhibits DA formation by modulating the reaction rate of TH to reduce the Ca(2+)-mediated phosphorylation levels of the TH molecule.

Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.

Homospecific activity (activity per enzyme protein) of tyrosine hydroxylase increases in parkinsonian brain

Tyrosine hydroxylase (TH) contents in the caudate nucleus, putamen, and substantia nigra from control and parkinsonism brains were measured for the first time by a sandwich enzyme immunoassay. Both the TH protein content and TH activity (Vmax) were decreased in parallel in the parkinsonian brains as compared with those of the control brains. In contrast, TH “homospecific activity” (activity per enzyme protein) was significantly increased in the parkinsonian brains. The results indicate that the decrease of TH activity in parkinsonian brains is due to the decrease of TH protein content as a result of cell death. The increase in the “homospecific activity” of residual TH in parkinsonian brain suggests such molecular changes in TH molecules as result in a compensatory increase in TH activity.

Acute effects of 1-methyl-4-phenylpyridinium ion (MPP+) on dopamine and serotonin metabolism in rat striatum as assayed in vivo by a micro-dialysis technique

The acute effect of 1-methyl-4-phenylpyridinium ion (MPP+), a neurotoxin derived from 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), was examined by the in vivo micro-dialysis technique. A dialysis cannula was implanted into rat striatum, and the changes in the concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in the perfusate every 20 min after administration of MPP+ were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ED). After MPP+ administration the levels of DOPAC, HVA and 5-HIAA were markedly decreased. On the contrary the level of DA was markedly increased and reached a maximum 40 min after beginning of the MPP+ administration. By postmortem analysis of the striatal tissue MPP+ was proved to cause the inhibition of monoamine oxidase (MAO), especially MAO-B. These results suggest that the acute biochemical changes induced by MPP+ in vivo were MAO inhibition and release of DA.

Effects of repeated systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal tyrosine hydroxylase activity in vitro and tyrosine hydroxylase content

We examined both in vitro tyrosine hydroxylase (TH) activity and TH content determined by a new enzyme immunoassay in the mouse striatum after repeated systemic injection of MPTP. Repeated systemic administration of MPTP to mice (30 mg/kg per day, subcutaneously for 8 days) caused an approximately 65% decrease of both TH activity and TH content in the striatum. The intensity of immunohistochemical staining of TH protein in the striatum was also reduced in MPTP-treated mice. These results indicate that the reduction of TH activity in vitro after the repeated administration of MPTP is due to reduction of TH protein as a result of nerve degeneration.

A selective Ca2+/calmodulin-dependent protein kinase II inhibitor, KN-62, inhibits the enhanced phosphorylation and the activation of tyrosine hydroxylase by 56 mM K+ in rat pheochromocytoma PC12h cells.

Involvement of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-kinase II) on the phosphorylation of tyrosine hydroxylase (TH, EC.1.14.16.2) in rat pheochromocytoma, PC12h cells was examined using KN-62, 1-[N,O-Bis(5-isoquinolinsulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipe razine, a selective inhibitor of Ca2+/CaM-kinase II. Both the enhanced phosphorylation of TH and the activated L-3,4-dihydroxyphenylalanine (DOPA) formation in the high K+ depolarization were inhibited by 10 microM KN-62. After incubation of PC12h cells with 10 microM KN-62 for 1 hr, the activation of TH with 3 min incubation of 56 mM K+ was reduced to the basal activity. However, KN-62 did not directly affect the activity of purified rat TH at pH 6.0 or 7.0. These results indicate that Ca2+/CaM-kinase II phosphorylates and activates TH of PC12h cells in the high K+ depolarization.

Expression of four types of human tyrosine hydroxylase in COS cells

Alternative splicing from a single gene produces four kinds of human tyrosine hydroxylase (types 1–4) which have structural diversity only in the N‐terminal region. We attempted expression of the type 1–4 enzymes in COS cells and performed kinetic analyses. All had enzymatic activities. The K m values of the four types for L‐tyrosine and 6‐methyl‐5,6,7,8‐tetrahydropteridine were similar, although their relative homospecific activities were clearly different. The type 1 enzyme displayed the highest activity.

Effects of Systemic Administration of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine to Mice on Tyrosine Hydroxylase, l-3,4-Dihydroxyphenylalanine Decarboxylase, Dopamine β-Hydroxylase, and Monoamine Oxidase Activities in the Striatum and Hypothalamus

Abstract: The effects of 1‐methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine (MPTP) (30 mg/kg subcutaneously per day for 8 days) to C57BL/6N mice were studied on tyrosine hydroxylase (TH), l‐3,4‐dihydroxyphenylalanine decarboxylase (DDC), and monoamine oxidase (MAO) activities in the striatum, and TH, DDC, dopamine‐β‐hydroxylase (DBH), and MAO activities in the hypothalamus. Treatment with MPTP led to a large decrease in TH activity and a parallel decrease in DDC activity in the striatum, as compared with the saline controls. In contrast, MPTP administration did not cause a decrease of the activities of TH, DDC, and DBH in the hypothalamus. There was also no reduction in MAO activities of striatum and hypothalamus. These data indicate that MPTP administration to mice results in specific degeneration of the dopaminergic nigrostriatal pathway and that DDC in the mouse striatum may mainly be localized in the dopaminergic neurons with TH.

Effects of myosin light-chain kinase inhibitor on catecholamine secretion from rat pheochromocytoma PC12h cells.

Release of dopamine from rat pheochromocytoma PC12h cells by high K+ (50 mM) was inhibited by a specific inhibitor of myosin light-chain kinase (ML-9) dose-dependently. The myosin light-chain kinase inhibitor also specifically inhibited the phosphorylation of a 20 KDa protein by myosin light-chain kinase. Myosin light chain kinase may play a stimulatory role in the release reaction of catecholamines from the rat pheochromocytoma cells.

Inactivation of tyrosine hydroxylase in rat striatum by 1-methyl-4-phenylpyridinium ion (MPP+)

We report that 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inactivated tyrosine hydroxylase (TH) when MPP+ was directly infused into the striatum. We examined both in vitro TH activity and TH content measured by an enzyme immunoassay in the rat striatum after MPP+ was administered by an in vivo brain microdialysis probe. MPP+ caused the inhibition of TH activity but did not influence TH content in the ipsilateral striatum. These results indicate that MPP+ may cause an acute inactivation of TH after continuous exposure at the high concentrations.