Masahiro Suno

Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition

Lipid peroxidation in rat brain mitochondria was induced by NADH in the presence of ADP and FeCl3. CV-2619 inhibited the lipid peroxidation in a concentration-dependent manner; the concentration giving 50% inhibition (IC50) was 84 microM. In addition, the inhibitory effect of CV-2619 was strongly enhanced by adding substrates of mitochondrial respiration; when succinate, glutamate, or succinate plus glutamate was added, the IC50 of CV-2619 was changed to 1.1, 10, or 0.5 microM, respectively. Metabolites of CV-2619 also inhibited the lipid peroxidation. The inhibitory effect of CV-2619 on mitochondrial lipid peroxidation disappeared when TTFA, an inhibitor of complex II in mitochondrial respiratory chain, was added. The results indicate that in mitochondria CV-2619 is changed to its reduced form which inhibits lipid peroxidation.

Autoradiographic distribution of pituitary adenylate cyclase activating polypeptide (PACAP) binding sites in the rat brain

Distribution of pituitary adenylate cyclase activating polypeptide (PACAP) binding sites was investigated in the rat brain and pituitary gland by means of in vitro autoradiography. High densities of specific [125I]PACAP binding were observed in the anterior pituitary, hippocampus (CA1-4 and dentate gyrus) and in the superior colliculus. Moderate to high labeling was observed in the periaqueductal gray matter, substantia nigra pars compacta, and in the habenula. The hypothalamus, thalamus, ventral tegmental area (VTA), mammillary body and medial geniculate body were moderately labeled. The present results support possible actions of PACAP on the pituitary functions, and further suggest that PACAP is a neurotransmitter/neuromodulator in the central nervous system.

Effects of idebenone on neurological deficits, local cerebral blood flow, and energy metabolism in rats with experimental cerebral ischemia

Improvement of energy metabolism in ischemic cerebral tissue benefits the therapy of occlusive cerebrovascular disorders. In the present study, the effects of 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (idebenone) on neurological signs, such as ischemic seizures, lactate and ATP contents of the cerebral cortex, and local cerebral blood flow, were assessed in stroke-prone spontaneously hypertensive rats (SHRSP) with experimentally induced cerebral ischemia. Experimental cerebral ischemia was caused by bilateral carotid artery occlusion (BCAO) in male SHRSP (8-10 weeks old). Pretreatment with idebenone (10-100 mg/kg, p.o.) for 3 or 10 days delayed the onset of ischemic seizure (acute stroke) and prolonged survival time in SHRSP roughly in a dose-dependent manner. When the compound (100 mg/kg, i.p.) was given once 30 min after BCAO, it exerted similar ameliorating effects on the neurological deficits. When idebenone (100 mg/kg for 3 days) was given orally, it did not significantly inhibit the decrease in regional cerebral blood flow induced by BCAO. However, the same treatment markedly inhibited increases in the lactate content and lactate/pyruvate ratio and the decrease in ATP content of the cerebral cortex. The compound did not affect cerebral blood flow in normal rats. These results suggest that idebenone ameliorates the neurological deficits related to cerebral ischemia, and that this effect is mediated by improving cerebral energy metabolism.

Recombinant human basic fibroblast growth factor (rhbFGF) induces secretion of nerve growth factor (NGF) in cultured rat astroglial cells

Recombinant human basic fibroblast growth factor (rhbFGF) was found to induce secretion of nerve growth factor (NGF) in cultured astrocytes from rat brain. The induction was concentration dependent; a maximum increase of 2-fold was observed at concentrations of 3-10 ng/ml or rhbFGF. When rhbFGF was added to the astrocytes, the induction occurred within 8 h and leveled off after 12 h. Antiserum against rhbFGF completely blocked the induction. Maximum induction occurred under serum-free culture conditions and in cultures with a high cell density. These results suggest that rhbFGF may exert a neurotrophic effect on the central nervous system through the induction of NGF secretion by astrocytes.

Inhibition of lipid peroxidation by idebenone in brain mitochondria in the presence of succinate

Lipid peroxidation in brain mitochondria was induced by NADH in the presence of ADP and FeCl3. A novel quinone compound, idebenone, inhibited this peroxidation and the inhibition was markedly enhanced by succinate, a substrate of mitochondrial respiration. The concentration of succinate required to exert the maximal effect was 1.5 mM. The concentration of idebenone giving 50% inhibition (IC50) was 0.5 and 84 microM in the presence and absence of succinate, respectively, indicating that succinate enhances the inhibition by 170-fold. Moreover, the inhibitory effect of idebenone in the presence of succinate was abolished by adding thenoyltrifluoroacetate (TTFA), an inhibitor of complex II in the mitochondrial respiratory chain. These results indicate that idebenone is changed through complex II to its reduced form, which protects mitochondria against lipid peroxidation.

Mechanism of increased sensitivity to cerebral ischemia following carotid artery occlusion in stroke-prone spontaneously hypertensive rats: importance of genetic factors.

Neurological symptoms and cerebral metabolism following bilateral carotid artery occlusion (BCAO) were observed in stroke-prone spontaneously hypertensive rats (SHRSP), stroke-resistant SHR (SHRSR), normal Wistar-Kyoto rats (WKY) and the F1 and F2 hybrids between SHRSP and SHRSR or WKY. Systolic blood pressure recorded before BCAO was found to rank in the following order: SHRSP greater than F1 (SHRSP X SHRSR) greater than SHRSR greater than F1 (SHRSP X WKY) greater than WKY. The effect of BCAO in these rats tended to be proportional to the blood pressure. F1 (SHRSP X WKY) was more sensitive to brain ischemia than SHRSR. In addition, though none of the SHRSR (average blood pressure 155 mm Hg) developed acute stroke symptoms, many animals of the F2 generation, in which the blood pressure was equal to or lower than that of SHRSR, developed stroke symptoms. Lactate and ATP changes in the F2 generations did not correlate with the blood pressure. The results suggest that genetic factors may play an important ro...

Inhibition of brain mitochondrial swelling by idebenone

The effects of idebenone on the swelling of rat brain mitochondria were studied. When FeCl3 was added to a mitochondrial suspension, a pronounced mitochondrial swelling occurred accompanied by the production of lipid peroxide; the two phenomena were closely correlated (r = 0.96, p less than 0.01). Idebenone inhibited the mitochondrial swelling and lipid peroxidation in a concentration-dependent manner; the concentration giving 50% inhibition was 37 microM for swelling and 53 microM for lipid peroxidation. Metabolites of idebenone also inhibited the lipid peroxidation. These results suggest that idebenone stabilizes the mitochondrial membrane by inhibiting lipid peroxidation in brain mitochondria.

Inhibition of platelet aggregation by idebenone and the mechanism of the inhibition

The inhibitory effect of 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (idebenone) on platelet aggregation was studied in rat and human platelets in vitro, and the mechanism of inhibition was examined in rat platelets. Idebenone inhibited the aggregation induced by collagen and thrombin in washed platelets, and by arachidonate and ADP in platelet-rich plasma (PRP). The inhibition was more prominent in collagen- and arachidonate-induced aggregation. In collagen-induced aggregation of human platelets, idebenone was 8-fold more potent than aspirin. In addition, idebenone inhibited prostaglandin synthesis and thromboxane B2 production, and also increased the cyclic AMP content in platelets. However, the concentration of idebenone required to inhibit thromboxane B2 production was much lower than that required to increase cyclic AMP. These results indicate that idebenone inhibits platelet aggregation by inhibiting thromboxane B2 synthesis rather than by increasing cyclic AMP content.

Effects of idebenone on lipid peroxidation and hemolysis in erythrocytes of stroke-prone spontaneously hypertensive rats

Stroke-prone spontaneously hypertensive rats (SHRSP) were kept on a 1% NaCl solution as drinking water to shorten the onset-time of a stroke. The level of lipoperoxide (LPO) in the erythrocytes of SHRSP loaded with salt for 22 days was significantly higher than that of the controls. Idebenone treatment (30 mg/kg per day, p.o.) markedly decreased the LPO to the level of the controls. Hemolysis in SHRSP was accelerated by the salt-loading. Idebenone significantly inhibited the hemolysis in a dose-dependent manner. These results suggest that idebenone inhibits lipid peroxidation in erythrocytes and stabilizes the erythrocyte membrane.

Rat prostatic growth factors : purification and characterization of high and low molecular weight epidermal growth factors from rat dorsolateral prostate

Growth factors which possibly participate in androgen-induced proliferation of rat prostate epithelial cells have been purified and characterized. Four distinct forms of growth factor were found in the extract of rat dorsolateral prostate. One of the factors was a member of heparin-binding growth factor (HBGF) family judging from its high affinity for heparin-Sepharose. The other three factors were capable of competing with [125I]epidermal growth factor (EGF) for the cell surface receptor, and recognized by anti-rat EGF antiserum. These EGF-like factors (EGF1-EGF3) were purified by ion-exchange chromatography, gel filtration and reverse phase HPLC. EGF1 showed microheterogeneity on chromatographic and electrophoretic separation and N-terminal sequence analysis. EGF1 showed an average molecular weight of about 35,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. These results indicated that EGF1 was a mixture of high molecular weight forms of EGF. The molecular weights of EGF2 and EGF3 were similar to that of rat submaxillary gland EGF (Mr = 5400). The amino acid sequence of EGF2 was identical with that of rat EGF except for the N- and C-terminal amino acids: aspartic acid instead of asparagine was found at the N-terminal position and C-terminal arginine was missing in EGF2. Although the N-terminal sequence of EGF3 (1-19) was identical with that of EGF2, the two factors were completely separated by gel filtration indicating a difference in the C-terminal structure. EGF1, EGF2 and EGF3 but not HBGF stimulated proliferation of primary cultured rat dorsolateral prostate epithelial cells.