Shinichi Hatta

Ion channels and diseases

 Ion channels play important roles in vital cellular signaling processes in both excitable and nonexcitable cells. Since 1987, a large number of channel genes have been cloned, and their biophysical properties, subunit stoichiometries, channel assemblies, and modulation by second messengers and ligands have been gradually elucidated. At present, more than ten ion channel genes have been identified as causing human hereditary diseases. Molecular techniques such as the positional cloning method are indispensable for finding new genes for channel-related diseases. Ion channels participate in the excitation-restoration of neurons and myocytes. Mutations of ion channels in these cells cause abnormal excitation and diseases such as long QT syndrome and ataxia. The second physiological function of ion channels, in addition to their regulation of cell excitability, is ion transport. Bartters syndrome and Liddles syndrome are due to abnormalities of ion transport. Most of these ion channel diseases are caused by loss of function, although some mutations are known to result in gain of function. The number of identified channel-related diseases is growing rapidly. Elucidation of the molecular basis of an ion channel disease not only provides new opportunities for early diagnosis and therapy for the disease but also provides clues to determine a previously unknown function of the ion channel.

Effects of bilobalide on γ-aminobutyric acid levels and glutamic acid decarboxylase in mouse brain

We have previously demonstrated that bilobalide, a constituent of the Ginkgo biloba extract, possesses anticonvulsant activity, and suggested that the mechanism of its anticonvulsant action involves modulation of y-aminobutyric acid (GABA)-related neuronal transmission. This study examined the effects of bilobalide on the level of GABA and glutamate, the activity and the amount of glutamic acid decarboxylase (EC 4.1.1.15), and the function of GABA(A) receptors in the hippocampus, cerebral cortex and striatum of the mouse. GABA levels, glutamic acid decarboxylase activity, and the protein amount of 67 kDa glutamic acid decarboxylase in the hippocampus of mice treated with bilobalide (30 mg/kg, p.o., once a day for 4 days) were significantly higher than those in controls. However, there were no significant differences in glutamate levels or, the number and the dissociation constants of GABA(A) receptors in the hippocampus between control and bilobalide-treated mice. These results suggest that the anticonvulsant effect of bilobalide is due to elevation of GABA levels, possibly through potentiation of glutamic acid decarboxylase activity and enhancement of the protein amount of 67 kDa glutamic acid decarboxylase by bilobalide.

Effects of extract of Ginkgo biloba leaves and its constituents on carcinogen-metabolizing enzyme activities and glutathione levels in mouse liver

The effects of a standardized extract of Ginkgo biloba L. leaves (EGb) and its terpene constituents, bilobalide and ginkgolides, on the activities of detoxification enzymes, i.e., glutathione S-transferases (GSTs) and DT-diaphorase, and glutathione contents, were investigated in the mouse liver. Oral treatment with EGb (100-1,000 mg/kg) and bilobalide (10-30 mg/kg) once a day for 4 days caused a dose-dependent elevation in GST activity. Ginkgolide A (30 mg/kg, for 4 days) also significantly elevated GST activity, whereas ginkgolide B and ginkgolide C at the same dose had no effects. EGb significantly increased the protein level of GST pi, and bilobalide significantly increased those of GST alpha and GST mu Moreover, EGb-treatment and bilobalide-treatment caused significant elevations in DT-diaphorase activity and in hepatic glutathione contents.

Ca2+/CaM-sensitive adenylyl cyclase activity is decreased in the Alzheimer's brain: possible relation to type I adenylyl cyclase.

SummaryImmunoreactivities of four subtypes of adenylyl cyclase (AC) (types I, II, IV and V/VI), and basal, forskolin- and Mn2+-stimulated AC activities with or without calcium and calmodulin (Ca2+/CaM) were estimated in parietal cortex membranes from cases with dementia of the Alzheimer type (DAT) and age-matched controls. Immunoreactivities of AC-I and AC-II were significantly decreased, but those of AC-IV and AC-V/VI did not change in DAT brains. There was a significant correlation of AC-I immunoreactivity with Ca2+/CaM-sensitive AC activity, but not with the Ca2+/CaM-insensitive activity. Ca2+/CaM-sensitive AC activity was significantly lower in DAT than in the control, indicating that impairment of Ca2+/CaM-sensitive AC-I is clearly involved in the pathophysiology of DAT.

Alterations of guanine nucleotide-binding proteins in post-mortem human brain in alcoholics.

Qualitative and quantitative alterations of G proteins in membrane preparations from parietal and temporal cortex regions in post-mortem brains obtained from alcoholics and controls matched with respect to age and post-mortem delay were investigated by Western-blotting with polyclonal antibodies against specific G protein subunits and functional photoaffinity GTP binding. Quantitative immunoblotting showed that only Gs alpha (52 kDa species) in temporal cortex was significantly decreased (30%, P < 0.05) in alcoholics compared with controls. Moreover, ethanol-stimulated photoaffinity GTP labeling of Gs alpha and Gi/o alpha was decreased in alcoholics in both cortex regions. These results suggest that disturbances of G protein-mediated signal transduction may be involved in the pathophysiology of alcoholics.

Participation of Tubulin in the Stimulatory Regulation of Adenylyl Cyclase in Rat Cerebral Cortex Membranes

Abstract: This study examined effects of tubulin on the activation of adenylyl cyclase in rat cerebral cortex membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis‐resistant GTP analogue 5′‐guanylylimidodiphosphate (GppNHp) caused significant activation of the enzyme by ∼156% under conditions in which stimulation rather than inhibition of the enzyme was favored. Tubulin‐GppNHp activated isoproterenol‐sensitive adenylyl cyclase, potentiated forskolin‐stimulated activity of the enzyme, and reduced agonist binding affinity for β‐adrenergic receptors. When tubulin, polymerized with the hydrolysis‐resistant photoaffinity GTP analogue [32P]P3(4‐azidoanilido)‐P1‐5′‐GTP ([32P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to Gsα as well as Giα. These results suggest that, in rat cerebral cortex membranes, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to Gsα, as well as affecting the Gi‐mediated inhibitory pathway.

Platelet GTP-binding protein in long-term abstinent alcoholics with an alcoholic first-degree relative

alcoholic patients had a family history of alcohol dependence. Ten subjects had an alcoholic first-degree relative (FLIP group; subjects had alcoholic fathers and/or siblings) and the other 10 had no first-degree or second-degree alcoholic relative (FHN group). The duration of alcohol use in the FHP and FHN group was 36.3 -+ t.33 and 34.9 +2.29 yea-s, respectively. Control subjects were agematched and gender*matched social drinkers drawn primarily from the faculty and staff of the Sapporo Medical University Hospital. The level of social drinking was determined by the Kurihama Alcoholism Screening Test (KAST), whereby a score of 2 or greater indicates a problem drinker. In addition to the KAST, both teetotalers and problem drinkers were excluded. The study procedures were fully explained and informed consent was obtained.

Bilobalide prevents reduction of γ-aminobutyric acid levels and glutamic acid decarboxylase activity induced by 4-O-methylpyridoxine in mouse hippocampus

We previously reported that bilobalide, a constituent of Ginkgo biloba L. leaves, protected mice against convulsions induced by 4-O-methylpyridoxine (MPN). To elucidate the mechanism of the anticonvulsant activity of bilobalide, this study examined the effect of bilobalide on MPN-induced changes in the levels of gamma-aminobutyric acid (GABA) and glutamate, and in the activity of glutamic acid decarboxylase (GAD) in the hippocampus, cerebral cortex and striatum of the mouse. GABA levels and GAD activity in the hippocampus and cerebral cortex were significantly enhanced by bilobalide treatment (30 mg/kg, p.o., for 4 days) alone. MPN significantly decreased GABA levels and GAD activity in the three brain regions tested compared with those in the control. Pretreatment with bilobalide effectively suppressed the MPN-induced reduction in GABA levels and GAD activity in the hippocampus and cerebral cortex. On the other hand, there were no significant differences in the glutamate levels in the three regions despite various treatments. These results suggested that bilobalide prevents MPN-induced reduction in GABA levels through potentiation by bilobalide of GAD activity, and this effect of bilobalide contributes to its anticonvulsant effect against MPN-induced convulsions.

Alterations of Tubulin Function Caused by Chronic Antidepressant Treatment in Rat Brain

Abstract1. Antidepressants have been used clinically for many years; however, the neurochemical mechanism for their therapeutic effect has not been clarified yet. Recent reports indicate that chronic antidepressant treatment directly affects the postsynaptic membrane to increase the coupling between the stimulatory GTP-binding (G) protein, Gs, and adenylyl cyclase. Tubulin, a cytoskeletal element, is involved in the stimulatory and inhibitory regulation of adenylyl cyclase in rat cerebral cortex via direct transfer of GTP to G proteins. In this study, we investigated whether the functional change of the adenylyl cyclase system caused by chronic antidepressant treatment involves an alteration of tubulin function in the regulation of adenylyl cyclase activity.2. Male Sprague–Dawley rats were treated once daily with amitriptyline or saline by intraperitoneal injection (10 mg/kg) for 21 days, and their cerebral cortex membranes and GppNHp-liganded tubulin (tubulin-GppNHp) were prepared for what.3. GppNHp-stimulated adenylyl cyclase activity in cortex membranes from amitriptyline-treated rats was significantly higher than that in control membranes. Furthermore, tubulin–GppNHp prepared from amitriptyline-treated rats was more potent than that from control rats in the stimulation of adenylyl cyclase activity in the cortex membranes of the controls. However, there was no significant difference in manganese-stimulated adenylyl cyclase activity between control and amitriptyline-treated rats.4. The present results suggest that chronic antidepressant treatment enhances not only the coupling between Gs and the catalytic subunit of adenylyl cyclase but also tubulin interaction with Gs in the cerebral cortex of the rat.

Diazepam physical dependence and withdrawal in rats is associated with alteration in GABAA receptor function.

Alteration in the function of the GABAA receptor complex and its relation to changes in withdrawal signs in diazepam (DZP)-dependent rats were studied. Physical dependence on DZP was induced in male F344 rats by using the drug-admixed food method. After cessation of treatment, withdrawal signs such as spontaneous convulsions were observed and withdrawal scores were maximal at 39 approximately 45 hr after the DZP withdrawal. Furthermore, these withdrawal signs almost disappeared by 159 approximately 168 hr after the DZP withdrawal. GABA-stimulated 36Cl- influx into cerebral cortical membrane vesicles was significantly decreased in rats 0 hr after DZP withdrawal and significantly increased in rats 42 hr after DZP withdrawal compared with control rats Flunitrazepam (FZ)-induced potentiation and an antagonistic effect of Ro 15-1788 on GABA-stimulated 36Cl- influx were observed in control rats. No FZ-potentiated GABA-stimulated 36Cl- influx was observed in rats 0 hr after DZP withdrawal: however, such an effect of FZ was recognized in rats 42 hr and 162 hr after DZP withdrawal. No antagonistic effect of Ro15-1788 on the FZ-induced stimulation was recognized in rats 0 hr and 42 hr after DZP withdrawal but was recognized at 162 hr after DZP treatment, although it was not significant. In a [3H]FZ assay of binding to benzodiazepine (BZ) receptors. Bmax values were significantly decreased in rats 0 hr after DZP withdrawal, but increased at 42 hr after DZP withdrawal, compared with control rats Bmax had almost returned to the control level at 162 hr after DZP treatment rats. In conclusion, these results indicate that functional changes in the GABAA/BZ receptor/CI- channel complex, i.e. increased sensitivity in GABAA receptors and impairment in the functional coupling between BZ receptors and GABAA receptors, may possibly be involved in the biochemical mechanism of the severe withdrawal symptoms appearing after chronic treatment with DZP.