Tadayoshi Hata

Targeted Disruption of the Tyrosine Hydroxylase Locus Results in Severe Catecholamine Depletion and Perinatal Lethality in Mice

Tyrosine 3-hydroxylase (TH, EC 1.14.16.2) catalyzes the first and rate-limiting step of the catecholamine biosynthetic pathway in the nervous and endocrine systems. The TH locus was disrupted in mouse embryonic stem cells by homologous recombination. Mice heterozygous for the TH mutation were apparently normal. In these mice, TH activity in the embryos and adult tissues was less than 50% of the wild-type values, but the catecholamine level was decreased only moderately in the developing animals and was maintained normally at adulthood, suggesting the presence of a regulatory mechanism for ensuring the proper catecholamine level during animal development. In contrast, the homozygous mutant mice died at a late stage of embryonic development or shortly after birth. Both TH mRNA and enzyme activity were lacking in the homozygous mutants, which thus explained the severe depletion of catecholamines. These changes, however, did not affect gross morphological development of the cells that normally express high catecholamine levels. Analysis of electrocardiograms of surviving newborn mutants showed bradycardia, suggesting an alteration of cardiac functions in the homozygous mice that may lead to the lethality of this mutation. In addition, transfer of a human TH transgene into the homozygous mice corrected the mutant phenotype, showing recovery of TH activity by expression of the human enzyme. These results indicate that TH is essential for survival of the animals during the late gestational development and after birth.

Viremia and neutralizing antibody response in infants with exanthem subitum

Mononuclear cell-associated viremia caused by human herpesvirus type 6 was detected in 39 (66%) of 59 blood samples from 38 children with exanthem subitum between day 0 and day 7 of the disease. The rate of virus isolation from mononuclear cells was 100% (26/26) on days 0 to 2 (just before appearance of skin rash), 82% (9/11) on day 3, 20% (2/10) on day 4, 7% (2/12) on days 5 to 7, and 0% (0/37) on day 8 and thereafter. The cell-free virus was detected in blood in 10 (21%) of 47 blood samples during the same period. The antibody activity to the virus, evaluated by a newly developed neutralization assay, was first detected on day 3 of the disease with a positive rate of 18% (2/11). It became 60% (6/10) on day 4, 75% (9/12) on days 5 to 7, and 100% on day 8 and thereafter. Thus the disappearance of the virus from blood was associated with the induction of specific immunity to the virus.

The role of Ca2+ release from sarcoplasmic reticulum in the regulation of sinoatrial node automaticity

SummaryThe role of Ca2+ release channels in the sarcoplasmic reticulum in modulating physiological automaticity of the sinoatrial (SA) node was studied by recording transmembrane action potentials and membrane ionic currents in small preparations of the rabbit SA node. Ryanodine, which modifies the conductance and gating behavior of the Ca2+ release channels, was used to block Ca2+ release from the sarcoplasmic reticulum. Superfusion of 1-mM ryanodine decreased the spontaneous firing frequency as well as the maximal rate of depolarization of the SA, and these reductions reached a steady state within approximately 5min. The action potential recordings revealed that the latter part of diastolic depolarization was depressed and that the take-off potential became less negative. This suggested that the negative chronotropic effect of ryanodine resulted from the blockade of physiological Ca2+ release from the sarcoplasmic reticulum. In voltage clamp experiments, using double-microelectrode techniques, ryanodine did not markedly reduce the Ca2+ current (ICa) but decreased the delayed rectifying K+ current (IK), the steady-state inward current (Iss), and the hyperpolarization-activated inward current (Ih). These observations suggest that, even when the function of Ca2+ channels in the cell membrane is normally maintained, depression of Ca2+ release channels in the sarcoplasmic reticulum would prevent sufficient elevation of the Ca2+ concentration in SA node cells for the activation of various ionic currents, and, thus adversely affect the physiological automaticity of this primary cardiac pacemaker.

Motor and learning dysfunction during postnatal development in mice defective in dopamine neuronal transmission

Mice lacking expression of tyrosine hydroxylase (TH), the first and rate‐limiting enzyme of the catecholamine biosynthetic pathway, in dopaminergic neuronal cell types were generated by a transgenic rescue approach to clarify the role of dopamine signaling during postnatal development. Introduction of the TH transgene directed by the dopamine β‐hydroxylase gene promoter into TH knockout mice restored noradrenaline and adrenaline synthesis, preventing perinatal lethality and cardiac dysfunction in the knockout mice. Lack of TH expression in the cells that normally express the dopaminergic phenotype resulted in a marked reduction of dopamine accumulation in the tissues, which led to multiple behavioral abnormalities at the juvenile stage. These abnormalities were characterized by a reduction in spontaneous locomotor activity, blockade of methamphetamine‐induced hyperactivity, cataleptic behavior, and defects in active avoidance learning. In contrast, development of the pituitary gland as well as production and secretion of the pituitary peptide hormones dependent on hypothalamic dopaminergic control were normally maintained, despite defective dopamine synthesis. These results demonstrate that dopamine neurotransmission is essential for controlling spontaneous and voluntary movement and associative learning during postnatal development through the nigrostriatal and mesocorticolimbic pathways. J. Neurosci. Res. 54: 450–464, 1998.

Autonomic neuropathy in transgenic mice caused by immunotoxin targeting of the peripheral nervous system.

Autonomic neuropathy in several neurodegenerative disorders results from disturbance in physiological functions of different cell types in the central and peripheral nervous systems. For a clearer understanding of the etiology and pathogenesis of the autonomic disorders it is necessary to create animal models in which degeneration of the causative neuronal types can be induced. Immunotoxin‐mediated cell targeting (IMCT) is a novel transgenic mouse technology for eliminating selective cell types with the cytotoxic activity of a recombinant immunotoxin anti‐Tac(Fv)‐PE40. In this study we conditionally disrupted peripheral catecholaminergic cells with IMCT to generate a mouse model developing autonomic failure based on primary defects of the sympathetic nervous system. Transgenic mice expressing human interleukin‐2 receptor α subunit under the control of the dopamine β‐hydroxylase gene promoter were intravenously treated with a proper dose of anti‐Tac(Fv)‐PE40. The immunotoxin induced a selective loss of the target cells in peripheral tissues of the transgenic mice and an impairment of catecholamine metabolism in the tissues. Targeting of the peripheral catecholaminergic cells resulted in severe and progressive phenotypic abnormalities mainly characterized by cardiac dysfunction, hypoactivity, and hypothermia, which explain development of autonomic neuropathy. Our IMCT strategy is useful for elucidating the involvement of different neuronal types and their interactions in the development and symptom of autonomic disorders. J. Neurosci. Res. 51:162–173, 1998.

A PDE3A mutation in familial hypertension and brachydactyly syndrome

Hypertension and brachydactyly syndrome (HTNB) with short stature is an autosomal-dominant disorder. Mutations in the PDE3A gene located at 12p12.2-p11.2 were recently identified in HTNB families. We found a novel heterozygous missense mutation c.1336T>C in exon 4 of the PDE3A gene in a Japanese family with multiple HTNB patients. This mutation was found to be completely linked to the family members who inherited this condition. The mutation, resulting in p.Ser446Pro, was located within the cluster region of reported mutations. This mutation may also affect the phosphodiesterase activity of PDE3A to reduce the cyclic AMP level in the cell and thereby influencing the development of limbs and the function of the cardiovascular system.

Electrophysiological effects of amrinone on the automaticity and membrane current system of the rabbit sinoatrial node cells

SummaryTo elucidate the physiological role of phosphodiesterase (PDE) in cardiac pacemaker cells, we studied the electrophysiological effects of amrinone, an inhibitor of PDE type III, on the spontaneous action potential (AP) and membrane currents, using small preparations (0.2 × 0.2 × 0.1mm) of rabbit sinoatrial (SA) node cells. Amrinone (0.1–1.0mM) progressively increased the AP amplitude, maximal rate of depolarization, and spontaneous firing frequency, shortened the AP duration, and made the threshold potential more negative. In voltage-clamp experiments using double microelectrode techniques, 0.1mM amrinone increased the Ca2+ current (ICa) obtained on step depolarization from −40 to −10mV by 25.86% ± 4.6% (P < 0.05,n = 6), the delayed rectifier K+ current (IK) tail obtained on repolarization from 10 to −60mV by 22.8% ± 4.7% (P < 0.05,n = 6), and the hyperpolarization-activated inward current (Ih) at −90mV by 19.5% ± 7.3% (P < 0.05,n = 6), respectively. Amrinone did not affect the slope factors of either the inactivation curve forICa (f∞ curve) or the activation curve for the delayed rectifierIK (p∞ curve). These results suggest that this PDE III inhibitor exerts a positive chronotropic action by enhancing the availability and the conductance of all the tested membrane currents in rabbit SA node cells.

Inflammation aggravates heterogeneity of ventricular repolarization in children with Kawasaki disease.

Kawasaki disease complicates with myocarditis and vasculitis. Even if myocarditis is asymptomatic, heterogeneity of ventricular repolarization may be increased in the acute phase. We evaluated whether the change in repolarization characteristics can be used as a predictor for myocarditis and coronary lesions. Enrolled 34 children who were treated with intravenous immunoglobulin therapy. There were no sequelae in the recovery phase in any subjects, including those who had transient coronary artery lesion. QT and the interval from the Tpeak to Tend (Tp-e) were determined. The Tp-e/QT ratios were compared between the acute and recovery phases and correlations with CRP level and body temperature were evaluated. A retrospective evaluation of Tp-e/QT as predictors of coronary dilation was also performed. Tp-e/QT in the acute phase correlated positively with body temperature and CRP level. In a comparison of patients with and without transient coronary artery lesion, Tp-e/QT was significantly higher in those with dilation. In conclusion, Tp-e/QT was strongly related to transient coronary dilation, in comparison with inflammatory indicators including fever and CRP level.

Dynamic QT/RR relationship of cardiac conduction in premature infants treated with low-dose doxapram hydrochloride.

Abstract Doxapram hydrochloride, a respiratory stimulant, has several undesirable side effects during high-dose administration, including second-degree atrioventricular (AV) block and QT prolongation. In Japan, this drug is contraindicated for newborn infants. Recent studies, however, have demonstrated the efficacy and safety of doxapram therapy for apnea of prematurity (AOP) using lower doses than those previously tested. As a result, approximately 60% of Japanese neonatologists continue to use this drug. This study used surface ECG recordings to assess the cardiac safety of low-dose doxapram hydrochloride (0.2 mg/kg/h) in fifteen premature very-low-birth-weight infants with idiopathic AOP. Cardiac intervals and number of apnea episodes were compared before and after drug administration. Low-dose doxapram hydrochloride resulted in approximately 90% reduction in the frequency of apnea without side effects. None of the infants developed QT or PR prolongation, arrhythmia, or other conduction disorders. In addition, there was no change in the slope of QT/RR before versus after administration of doxapram hydrochloride. We conclude that low-dose administration of doxapram hydrochloride did not have any undesirable effects on myocardial depolarization and repolarization.

Autonomic modulation of sinus and atrioventricular nodes in premature low-birth-weight infants

Respiratory vagal activity is expressed by heart rate variability (HRV) at approximately 1 month of age in premature low‐birth‐weight infants (PLBWI). However, the autonomic inputs into the sinus node (SAN) and atrioventricular node (AVN) in PLBWI are unclear. We evaluated the variability in PP and PR intervals at day zero (day 0) and 1 month (1 month) after birth in 16 PLBWI (gestation 32.3 ± 1.3 weeks, birth weight 1.578 ± 257 g). The polygraph was recorded during sleep on day 0 and at 1 month. PP and PR intervals and the number of respiratory cycles were measured, and frequency analysis was performed by auto‐correlation fast Fourier transforms. Power spectral density (PSD: ms2) was calculated for the low frequency domain (LF: 0.036∼0.146 Hz), high frequency domain (HF: 0.146∼0.390 Hz), total frequency (TF: 0.036∼2.000 Hz), and respiratory sinus arrhythmia (RSA: frequency bandwidth of 0.3 Hz with peak respiratory frequency as median), and the PSD ratio in the PP and PR intervals (LF/HF, RSA/TF) were compared. Compared with day 0, a decrease in the LF/HF ratio and an increase in the RSA/TF ratio in PP intervals were observed at 1 month, consistent with expression of respiratory vagal activity. For PR intervals, on the other hand, the LF/HF ratio increased, indicative of accentuated sympathetic activity. However, the respiratory vagal input was weak, and the RSA/TF ratio remained unchanged. These observations suggest that, in PLBWI at 1 month, AVN conduction was not predominatly influenced by respiratory‐related vagal activity, but was controlled by autonomic regulation, independent of the SAN.