Sumio Ohnishi

Uneven distribution of intracellular Cl− in rat hippocampal neurons

Electrophysiological observations of neurons suggest that perikarya and dendrites differ in local intracellular Cl- concentration ([Cl-]i), that has not been demonstrated yet. Regional [Cl-]i in cultured hippocampal neurons was estimated using a Cl(-)-sensitive fluorescent dye. Calibration showed that perikaryonic [Cl-]i was lower than dendritic [Cl-]i. Ethacrynic acid, an inhibitor of the outwardly directed Cl(-)-pump, increased the perikaryonic but not dendritic [Cl-]i. A decrease in [Cl-]i induced by furosemide or bumetanide, inhibitors of Na+/K+/2Cl- cotransporters, was more prominent in dendrites than in perikarya. These findings suggest that uneven distribution of Cl- is generated by the region-specific localization of these transporters.

An ATP-driven Cl− pump regulates Cl− concentrations in rat hippocampal neurons

To investigate the role of Cl(-)-stimulated Mg(2+)-ATPase (Cl(-)-ATPase) in neurons, we examined the effects of ethacrynic acid (0.3 mM), which completely inhibits Cl(-)-ATPase on the intracellular Cl- concentrations of cultured rat hippocampal neurons, using Cl(-)-sensitive fluorescent probes. Ethacrynic acid and ATP consuming treatment increased the intracellular Cl- concentration, but elevation of the extracellular K+ concentration up to 10 mM, inhibition of Na+/K(+)-ATPase, or dissolution of H+ gradients had no effect. Furosemide (0.1 mM), an inhibitor of Na+/K+/Cl- co-transport, decreased the intracellular Cl- concentrations. These results indicate that an ethacrynic acid-sensitive and ATP-driven Cl- pump functions to reduce intraneural Cl- concentrations.

Effects of Efferent Neurotransmitters on Intracellular Ca2+ Concentration in Vestibular Hair Cells of the Guinea Pig

The effects of putative efferent neurotransmitters on intracellular Ca2+ concentration ([Ca2+]i) in isolated vestibular hair cells (VHCs) of the guinea pig were determined using the Ca2+ sensitive dye Fura-2 and digital imaging microscopy. In the presence of 1 mM acetylcholine (ACh) there was a gradual increase in [Ca2+]i. In the presence of 10 microM ATP there was a rapid rise in [Ca2+]i. Thus, both ACh and ATP seem to be efferent neurotransmitters in VHCs of the guinea pig. When 100 microM Gamma-aminobutyric acid (GABA) was added there was a rapid increase in [Ca2+]i; thus GABA also seems to be an efferent neurotransmitter in VHCs. With the independent addition of 10 microM calcitonin gene-related peptide and 10 microM M- and L-enkephalin there were no significant increase in [Ca2+]i. We presume that these neuropeptides were functioning as efferent neuromodulators rather than as neurotransmitters.

Effects of protein kinase and phosphatase inhibitors on slow shortening of guinea pig cochlear outer hair cells.

The intracellular mechanisms of slow shortening in isolated guinea pig cochlear outer hair cells were investigated using inhibitors and/or an activator of protein kinases and protein phosphatases. The slow shortening was induced by tetanic electrical field stimulation, and changes in the cell length, volume and intracellular Cl- concentration were microscopically monitored using a chloride-sensitive fluorescent dye. The slow shortening was inhibited by a calmodulin inhibitor, W-7, and a calcium calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN-62. The inhibition by W-7 or KN-62, was abolished by the supplemented conductance of K+ with valinomycin. Among the protein phosphatase inhibitors tested, a type 1 and 2A protein phosphatase inhibitor, calyculin A, inhibited the slow shortening. The inhibition by calyculin A was abolished by the increased Cl- permeability, but neither by the increased K+ conductance with valinomycin nor by the increased Ca2+ conductance with A23187. A protein serine/threonine phosphatase activator, N-acetylsphingosine, inhibited the shortening, which was abolished by either valinomycin or a type 2A protein phosphatase inhibitor, okadaic acid, but not by calyculin A. These findings suggest the following signaling mechanisms in the slow shortening of outer hair cells; the K+ channel opening is facilitated through protein phosphorylation by CaMKII and suppressed via okadaic acid-sensitive dephosphorylation, and the Cl- channel opening depends on calyculin A-sensitive protein phosphatase activity.

Regulation of Cl− Conductance in Delayed Shortening and Shrinkage of Outer Hair Cells

Tetanic electrical field stimulation induces slow and delayed shortening of isolated outer hair cells (OHCs) from guinea pigs concomitant with a decrease in cell volume and intracellular Cl- concentration ([Cl-]i). These responses are dependent on the entry of Ca2+ and the activation of both Ca(2+)-activated K+ channels and furosemide-sensitive Cl- channels. In this study, the regulatory mechanisms of the Cl- channels of OHCs were investigated. Tetanic stimulation induced shrinkage and a decrease in [Cl-]i, the latter being unrelated to the activity of the Na+/K+/2Cl- cotransporter. These responses were inhibited by tetraethylammonium, and the inhibition was overcome by an increase in the K+ conductance by valinomycin, though valinomycin alone did not affect the resting cell volume and [Cl-]i. The deprivation of extracellular Ca2+ inhibited the stimulation-induced changes in volume and [Cl-]i even in the presence of valinomycin. These results suggest that electrical stimulation-induced delayed shortening and shrinkage of OHCs are induced by Cl- efflux, which is electrically coupled to K+ efflux and independently activated following an increase in intracellular Ca2+ concentration.

Ethacrynic acid-induced convulsions and brain noradrenaline in mice

The intracerebroventricular injection of ethacrynic acid (a 50% convulsive dose; 50 micrograms/mouse) accelerated brain noradrenaline turnover and decreased noradrenaline contents. The decrease in noradrenaline contents was antagonized by 2-amino-5-phosphonovalerate but not by diazepam. Both 2-amino-5-phosphonovalerate and diazepam suppressed the incidence of ethacrynic acid-induced convulsions while reserpine, alpha-methyl-para-tyrosine or FLA-63 augmented it. The results suggest that stimulation by ethacrynic acid of excitatory amino acid neurons enhances-noradrenergic neuronal anticonvulsive activity.

Squamous Cell Carcinoma in Thyroid Papillary Carcinoma.

We report the case of a 62-year-old man with a Squamous cell carcinoma of the thyroid gland which had invaded the hypopharynx. Primary squamous cell carcinoma of the thyroid gland is extremely rare. Its clinical course is very aggressive and the prognosis is poor. The patient underwent a radical resection of the thyroid, hypopharynx, larynx and resection of the trachea and esophagus was conducted concomitantly with bilateral neck dissection. Reconstruction of the terminal tracheostomy in the anterior mediastinum was created using a pectoralis major muscle flap.

Clinical Studies of Oral Cancer.

Seventy-three cases of oral cancer (tongue: 47, oral floor: 10, buccal mucosa: 10, gingiva: 4, hard palate: 2) treatedinitially from 1985 to 1995 were reviewed retrospectively. The cumulative five year survival rate was 71.4% for all of the patients, and 75.8% for carcinomas of the tongue, 88.9% for carcinomas of the buccal mucosa and 42.2% for carcinomas of the oral floor. Further strategies are required in advanced cases with T4 staging in order to improve the survival rate and quality of life of oral cancer patients.