Osamu Hatase

On the stabilization by fixation of configurational states in beef heart mitochondria

The energized configuration of the cristal membrane of beef heart mitochondria can be maintained only as long as oxygen is available for electron transfer. When the oxygen supply is exhausted, the membrane undergoes a transition to the nonenergized configuration. Since the exhaustion of the available oxygen supply is complete in 5–20 sec, it is impossible to apply the method of sedimenting the mitochondria prior to fixation for studying the energized configurational states of mitochondria. The direct addition of glutaraldehyde followed by osmium tetroxide to the mitochondrial suspension is the most effective way of freezing the configurational state of the cristal membrane. Fixation with glutaraldehyde appears to be complete within 1–2 sec even at 0°. Osmium tetroxide alone can also “freeze” the energized configuration by fixation but the concentration of the fixative is critical. The problem of capturing the configurational state applies not only to energized transitions (nonenergized to energized) but also to nonenergized transitions (orthodox to aggregated). The freezing by fixation of the cristal membrane in the aggregated configuration is best accomplished by the sequential use of glutaraldehyde and osmium tetroxide. When the levels of glutaraldehyde and osmium tetroxide are respectively too low or too high, the mitochondrion will undergo a transition from the aggregated to the orthodox configuration before fixation is complete. Light-scattering studies provide an independent method for monitoring configurational changes in mitochondria; these light-scattering measurements confirm that the conditions for fixation which lead to stabilization of the energized state as judged by electron microscopy, also show maintenance of configuration as judged by absence of light-scattering changes after the fixatives are introduced. Reagents used in negative staining will induce the geometrical form of the energized configuration of the mitochondrion even under nonenergizing conditions. These reagents are thus unsuitable for use in studies of configurational transitions in mitochondria.

A lattice structure in beef heart mitochondria induced by phosphotungstic acid.

Ordered arrays of structured material were visualized in the intracristal space of isolated beef heart mitochondria in two ways. Under standard conditions of fixation, structured material in the intracristal space appeared as paracrystalline arrays nestled between two apposing membranes. When mitochondria were preincubated with phosphotungstic acid (PTA) prior to fixation, the structures in the mitochondrial intracristal space took on an open lattice structure. Such structures, either paracrystalline or lattice, could not be demonstrated in the mitochondrial matrix space under these conditions.Pretreatment with PTA prior to fixation increased greatly the frequency with which structured material was observed within the mitochondrial intracristal space. Visualization of the PTA-induced lattice structures appeared to be pH dependent, being most clearly seen between pH 7·0 and 7·5. Above pH 7·5, lattice structures could not be seen, whereas at pH values below 7·0, the observed structures in the intracristal space no longer retained an organized lattice structure but became amorphous. Increasing the concentration of PTA from 0·1% to 3·5% or the incubation time from 5 sec to 1 h did not significantly alter the frequency of observation of lattice structures, as long as the mitochondrial preincubation with PTA was carried out between pH 7·0 and 7·5.

Specific inhibition of phosphate transport in mitochondria by N-ethylmaleimide

N-ethylmaleimide (NEM), a reagent that alkylates free sulfhydryl groups, was shown to be a highly effective inhibitor of the following coupled mitochondrial processes: oxidative phosphorylation, ATP-32Pi exchange, Pi-induced light scattering and configurational changes, State III respiration, valinomycin-induced translocation of potassium with Pi as the anion, and calcium accumulation in presence of Pi. However, NEM was less effective or ineffective in inhibiting some processes that do not require inorganic Pi, namely electron transfer and ATPase activity, ADP binding, energized light scattering changes induced by arsenate and nonenergized light scattering changes induced by acetate. The rate of oxidative phosphorylation and of ATP-32Pi exchange was normal in ETPH particles prepared from NEM-treated mitochondria. Also NEM, even et levels 2–3 times greater than those required to inhibit oxidative phosphorylation in intact mitochondria, did not inhibit coupled processes in submitochondrial particles. We are proposing that NEM alkylates sulfhydryl groups in the mitochondrion that modulate Pi translocation, and that the suppression of Pi translocation blocks oxidative phosphorylation, the Pi-dependent energized configurational change in mitochondria and Pi-dependent transport processes.

On the correlation of configurational changes in the inner mitochondrial membrane with energization.

Two reports in the literature that mitochondria isolated in special media or from a particular source do not undergo configurational changes under energizing, conditions have been analyzed in detail. It could be shown that the failure to demonstrate configurational changes was a consequence of a procedure which allowed anaerobiosis to set in before the mitochondria were fixed. When fixation was achieved while the mitochondria were still under energizing conditions, the correlation between configurational change and change in the energy state could be confirmed.

Treatment of Vasogenic Brain Edema by V1 Receptor Antagonist of Arginine Vasopressin

Centrally released arginine vasopressin (AVP) has been reported to increase the water permeability of brain capillaries under normal and pathological conditions. In present experiment, we divided cold-injured brain into cortical and deep structure, and studied the effect of central administration of V1 receptor antagonist (5 ng, 50 ng, and 500 ng per rat) on vasogenic brain edema. Cold injury induced significant increases in brain water and tissue sodium content of bilateral cortical structures, but no changes in bilateral deep structures. Intraventricular administration of Vi receptor antagonist (50 ng) significantly reduced this accumulation of water and sodium in cortical structures without any change in plasma osmolality. A large quantity of this antagonist (500 ng) showed no changes in brain water content of bilateral cortical structures. It is suggested that this antagonist has an optimal concentration of this antagonist for inhibiting vasogenic brain edema.

Analysis of Dopamine and Neuropeptides in 6-OHDA-Lesioned Rats

Cholecystokinin (CCK) and dopamine (DA) coexist in the ventral tegmental area and medial substantia nigra neurons.1 In the brain of Parkinson’s disease patients, a prominent decrease in CCK content of the substantia nigra has been reported,2 and it is thought that DA and CCK might be related to the etiology of Parkinson’s disease. The precise functional interaction between DA and CCK, however, is not clarified. Based on the hypothesis that CCK has a functional role as one of the modulators of DA metabolism, ceruletide (CLT), which is a CCK-related decapeptide, is currently under trial for the treatment of diseases with involuntary movements including Parkinson’s disease.3 In the present study we attempted to clarify the mechanism of action of CLT and CCK with respect to their receptors as well as their effect on behavior in a Parkinson’s disease model, namely, the 6-OHDA unilaterally lesioned rat.