Mitsunobu Matsubara

Prognostic significance of the nocturnal decline in blood pressure in individuals with and without high 24-h blood pressure: the Ohasama study.

Objective To examine the relationship between the normal nocturnal decline in blood pressure and the risk of cardiovascular mortality in individuals with and without high 24-h blood pressure values. Methods We obtained 24-h ambulatory blood pressure readings from 1542 residents of Ohasama, Japan, who were aged 40 years or more and were representative of the Japanese general population. We then followed up their survival for a mean of 9.2 years. The relationship was analysed using a Cox proportional hazards model adjusted for possible confounding factors. Results There was a linear relationship between the nocturnal decline in blood pressure and cardiovascular mortality. On average, each 5% decrease in the decline in nocturnal systolic/diastolic blood pressure was associated with an approximately 20% greater risk of cardiovascular mortality. There were no significant interactions for the risk between 24-h systolic/diastolic blood pressure values and continuous values for the nocturnal decline in blood pressure (P for interaction > 0.6). Even when 24-h blood pressure values were within the normal range (< 135/80 mmHg, average 118/69 mmHg), diminished nocturnal decreases in systolic/diastolic blood pressure were associated with an increased risk of cardiovascular mortality. Conclusions This is the first study to demonstrate that a diminished nocturnal decline in blood pressure is a risk factor for cardiovascular mortality, independent of the overall blood pressure load during a 24-h period, in the general population.

Prognostic significance of blood pressure and heart rate variabilities: the Ohasama study

To investigate the association between cardiovascular mortality and short-term variabilities in blood pressure and heart rate, we performed a long-term prospective study of ambulatory blood pressure monitoring in Ohasama, Japan, starting in 1987. We obtained ambulatory blood pressure and heart rate in 1542 subjects ≥40 years of age. Blood pressure and heart rate variabilities were estimated as a standard deviation measured every 30 minutes by ambulatory monitoring. There were 67 cardiovascular deaths during the follow-up period (mean=8.5 years). The Cox proportional hazards model, adjusted for possible confounding factors, demonstrated a significant increase in cardiovascular mortality, with an increase in daytime systolic ambulatory blood pressure variability. A similar trend was observed in daytime diastolic and nighttime ambulatory blood pressures. Cardiovascular mortality rate increased linearly, with a decrease in daytime heart rate variability. Subjects in whom the daytime systolic ambulatory blood pressure variability was larger than third quintile and the daytime heart rate variability was lower than the mean−SD were at extremely high risk of cardiovascular mortality. The blood pressure and heart rate variabilities obtained every 30 minutes by ambulatory blood pressure monitoring were independent predictors for cardiovascular mortality in the general population.

Usefulness of home blood pressure measurements in assessing the effect of treatment in a single-blind placebo-controlled open trial.

Objectives Reproducibility of home blood pressure measurements (HBP), and the placebo effect on HBP, were examined to establish the number of subjects required in order to observe a significant antihypertensive effect of a drug for a comparative study between placebo and active treatment. Methods Reproducibility of HBP (n = 172 for systolic blood pressure and n = 137 for diastolic blood pressure) and the placebo effect on HBP (n = 35 for systolic and n = 42 for diastolic blood pressure) were examined using a semi-automatic electronic device on untreated hypertensive subjects during 21 consecutive days of measurements. From these two studies, the number of subjects required in order to observe a significant antihypertensive drug effect was assessed. In both studies, measurements from the first 3 days were excluded from the analysis. Results Reproducibility, defined as the difference between the initial 5 day average (days 4–8) and the last 5 day average (days 17–21) was −1.9 ± 7.0/–1.4 ± 4.8 mmHg (mean ± SD), indicating high reproducibility with a minimal regression to the mean effect. A placebo was administered after the 8 day run-in period. The difference between the initial 5 day average (days 4–8) and the last 5 day average (days 17–21) was compared. The mean difference was 1.1 ± 6.2/0.2 ± 5.7 mmHg, suggesting that there was a minimal, if any, placebo effect. As determined by the power calculations, based on the standard deviations of the tests for reproducibility and the placebo effect, nine of 13 subjects are required to show that a 9/5 mmHg decrease in systolic HBP/diastolic HBP (50% of estimated effective decrease in systolic HBP/diastolic HBP according to the guidelines for clinical trials in Japan) is statistically significant. Conclusions The results suggest that the HBP measurement is highly reproducible and that there is a minimal, if any, placebo effect. These characteristics of HBP contribute to minimizing the number of subjects necessary for assessment of antihypertensive drug effects in comparative studies between an active treatment and placebo.

Expression of S‐100 protein is related to neuronal damage in MPTP‐treated mice

S‐100β is a calcium‐binding protein expressed at high levels in brain and is known as a marker of brain damage. However, little is known about the role of S‐100β protein during neuronal damage caused by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). To determine whether S‐100β protein is induced in glial cells after MPTP treatment, we investigated the expression of S‐100 protein immunohistochemically, using MPTP‐treated mice. We also examined the change of neurons and glial cells in mice after MPTP treatment. The present study shows that tyrosine hydroxylase (TH) immunoreactivity decreased gradually in the striatum and substantia nigra from 1 day after MPTP treatment. Thereafter, TH‐immunopositive cells and fibers decreased in the striatum and substantia nigra at 3 days after MPTP treatment. In contrast, S‐100‐immunopositive cells and glial fibrillary acidic protein (GFAP)‐immunopositive cells increased markedly in the striatum and substantia nigra at 3 days after MPTP treatment. Seven days after MPTP treatment, S‐100‐immunopositive cells decreased in the striatum and substantia nigra. However, the number of GFAP‐immunopositive cells increased in these regions. In double‐labeled immunostaining with anti‐S‐100 and anti‐GFAP antibodies, S‐100 immunoreactivity was observed only in the GFAP‐positive astrocytes. These results provide evidence that astrocytic activation may play a role in the pathogenesis of MPTP‐induced degeneration of dopaminergic neurons. Furthermore, the present study demonstrates that S‐100 protein is expressed selectively by astrocytes, but not by microglia, after MPTP treatment. These results provide valuable information for the pathogenesis of the acute stage of Parkinsons disease. GLIA 42:307–313, 2003.

Leucine-Rich Repeat-Containing G Protein-Coupled Receptor-4 (LGR4, Gpr48) Is Essential for Renal Development in Mice

Leucine-rich repeat-containing G protein-coupled receptor (LGR)-4 is a G protein-coupled receptor (GPCR) with a seven-transmembrane domain structure. LGRs are evolutionally and structurally phylogenetic, classified into three subgroups and are members of the so-called orphan receptors whose ligands have yet to be identified. We generated knockout mice lacking Lgr4(Gpr48) by targeted deletion of part of exon 18, which codes for the transmembrane and signal-transducing domains of the receptor. Lgr4 null mice were born at much less than the 25% expected frequency from crosses of Lgr4 heterozygous mice (Lgr4+/–). Lgr4 null mice that survived in utero died shortly after birth in almost all cases. We observed striking renal hypoplasia in the null mice, accompanied by elevated concentration of plasma creatinine. Histological analysis of the P0 null mouse kidney showed a notable decrease in the total number and density of the glomerulus. Thus, the function of Lgr4 is essential to regulate renal development in the mouse. This study suggests that the Lgr4 gene is a new and important member of LGRs involved in a group of genes responsible for hereditary disease in the kidney.

Device for the self-measurement of blood pressure that can monitor blood pressure during sleep.

Sponsorship: This work was supported by Research Grants from the Takeda Medical Foundation, the Japan Arteriosclerosis Prevention Fund ( ) ( ) JAPF, 2000 and the Mitsui Life Social Welfare Foundation 1998 , by Research Grants of Kosei Kagaku Kenkyuhi, 1996 and 1997, of Junkankibyo ( ) Itaku Kenkyuu 11C-5 , 1999, and Rojin Hoken Jigyo, 1998 and 1999, from the Ministry of Health and Welfare, and by a Research Grant for Scientific ( ) ( ) Research 10470102 and a Research Grant for JSPS’ Research T.O. from the Ministry of Education, Science and Culture of Japan.

alpha-Adducin Gly460Trp polymorphism is associated with low renin hypertension in younger subjects in the Ohasama study.

Background The Gly460Trp polymorphism of the alpha-adducin gene (ADD-1) has been examined as a candidate gene for essential hypertension with salt sensitivity in the Caucasian population. However, we failed to detect a positive association between the Gly460Trp polymorphism of ADD-1 and hypertension in a small series of Japanese subjects. Objective To examine the precise association between the Gly460Trp polymorphism of ADD-1 and blood pressure (BP), we carried out an association study using a Japanese population: the Ohasama Study. Design Subjects (n = 1490) were recruited from participants in the Ohasama Study, which is a cohort in a rural community of northern Japan. Methods DNA was extracted from the buffy coat of the participants who gave informed consent for genetic analysis, and the Gly460Trp polymorphism of ADD-1 was determined by the TaqMan polymerase chain reaction method. Various BP values (casual BP, ambulatory BP and home BP) were measured in the Ohasama study. We used the mean values of these BP measurements for analysis. Results The frequencies of genotypes in the Ohasama population were 23% Gly/Gly, 49% Gly/Trp, and 28% Trp/Trp. In the baseline characteristics, age, sex, body mass index, frequency of diabetes and hyperlipidemia were significantly different between hypertensive or normotensive subjects. In total subjects, all BP values were not different among ADD-1 genotypes. In the younger subjects (< 60 years old) with low plasma renin activity (< 1.0 ng/ml per h), however, ambulatory BP and home BP were significantly higher in the subjects with the Gly/Trp or Trp/Trp genotypes of ADD-1 polymorphism than in those with the Gly/Gly genotype. In the same population, the frequency of the Gly/Trp or Trp/Trp genotypes of ADD-1 was significantly higher in hypertensives than in normotensives (83 versus 72%, χ12 = 4.04, P < 0.05; odds ratio, 2.12; 95% confidence interval, 1.02–4.68). Conclusions These findings suggest the possibility that the Gly460Trp polymorphism of ADD-1 is associated with low renin hypertension.

Neuroprotective effect of riluzole in MPTP-treated mice

The neuroprotective effects of riluzole, a Na(+) channel blocker with antiglutamatergic activity, and MK-801, a blocker of N-methyl-D-aspartate (NMDA) receptors, were compared in the model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced depletion of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels in mice. The mice were injected intraperitoneally (i.p.) with four administrations of MPTP (10 mg/kg) at 1 h intervals and then the brains were analyzed 1, 3 and 7 days after the treatment. Dopamine and DOPAC levels were significantly decreased in the striatum from 1 day after MPTP treatment. A severe depletion in dopamine and DOPAC levels was found in the striatum 3 and 7 days after MPTP treatment. Riluzole antagonized the MPTP-induced decrease in dopamine, DOPAC and HVA levels in the striatum. On the other hand, MK-801 prevented the MPTP-induced decrease in DOPAC levels, but not in dopamine levels in the striatum. An immunohistochemical study indicated that riluzole can protect against MPTP-induced neuronal damage in the substantia nigra. These results suggest that riluzole is effective against MPTP-induced neurodegeneration of the nigrostriatal dopaminergic neuronal pathway.

Protective effects of neuronal nitric oxide synthase inhibitor in mouse brain against MPTP neurotoxicity: an immunohistological study

We recently reported that neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole, can protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice. It protected against both dopamine depletions and tyrosine hydroxylase (TH) positive neuron decreases in the mouse brain. In the present study, we further examined whether 7-nitroindazole can also protect against the alterations of TH-, microtubule-associated protein 2a,b (MAP2)-, glial fibrillary acidic protein (GFAP)-, parvalbumin (PV)-, dopamine transporter (DAT)-, nNOS- or endothelial NOS (eNOS)-positive cells, in comparison with pargyline as a relatively selective inhibitor of the monoamine oxidase-B (MAO-B). The present study showed that nNOS inhibitor as well as MAO-B inhibitor has a dose-dependent protective effect against MPTP-induced striatal dopamine and DOPAC depletion in mice. Furthermore, the present study revealed that 7-nitroindazole and pargyline can protect the alterations of immunohistochemical changes in the striatum and substantia nigra after MPTP treatment. These protective effects may be, at least in part, produced by the reduction of neuronally derived NO and peroxynitrite caused by MPTP. Our results also demonstrate that MPTP can cause functional damage of interneurons in the substantia nigra. These results suggest the possibility that nNOS inhibitors as well as MAO-B inhibitors may be therapeutically useful in neurodegenerative diseases such as Parkinsons disease. Thus our present results provide valuable information for the pathogenesis of degeneration of the nigrostriatal dopaminergic neuronal pathway.

Biochemical and immunohistological changes in the brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse

We investigated neurochemically and neuropathologically the utility of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice as a model of Parkinsons disease. The changes in dopamine D1 and D2 receptors and dopamine uptake sites were determined by quantitative autoradiography using [3H]SCH23390, [3H]raclopride and [3H]mazindol, respectively. Dopamine and 3,4-dihydroxyphenyl acetic acid (DOPAC) contents in the striatum were measured by high-performance liquid chromatography. The distribution of nigral neurons and reactive astrocytes was determined by immunohistochemical staining with antibody against tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP). The mice received four intraperitoneal injections of MPTP (10 mg/kg) at 1-h intervals and then the brains were analyzed at 3 and 7 days after the treatments. No significant change in dopamine D1 receptors was observed in the striatum and substantia nigra after acute treatment with MPTP. Dopamine D2 receptors were reduced significantly in the substantia nigra only 7 days after the MPTP treatment, whereas striatum showed no significant change in the binding throughout the experiments. In contrast, dopamine uptake sites were reduced markedly in the striatum and substantia nigra 3 and 7 days after the MPTP treatment. Dopamine and DOPAC content were also reduced in the striatum 3 and 7 days after the MPTP treatment. An immunohistochemical study indicated a loss of the number of TH-positive neurons in the substantia nigra 7 days after the MPTP treatment. In contrast, numerous GFAP-positive astrocytes were evident in the striatum 7 days after the MPTP treatment. These results provide valuable information for the pathogenesis of acute stage of Parkinsons disease.