|Budget Amount *help
¥11,500,000 (Direct Cost: ¥11,500,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1997: ¥7,900,000 (Direct Cost: ¥7,900,000)
In the previous studies using rat brain ischemia model, we found that nuclei of cerebral neurons contain high concentrations of pro μ-calpain and it is rapidly activated under the conditions of ischemia/hypoxia. In this study, we analyzed neuronal death induced by activation of pro μ-calpain and protective effects of calpain inhibitors and calcium chelate compounds using neuroblastoma cell line GOTO cells.
1. Distributions of calpain/calpastatin and influx of calcium ions. By immunohistochemistry, pro μ-calpain was densely stained in the nuclei of neurons, whereas calpastatin, endogenous inhibitor of calpain, was found in cytoplasma. By the use of ARGUS-50, intracellular calcium concentrations were estimated. The concentrations rapidly increased and reached plateau within 5 min under hypoxic conditions (1/5 O_2 partial preseure). The increasing calcium levels were always 10-20 % higher in nuclei than that of cytoplasma.
2. Neuronal death estimated by Trypan blue exclusion test. Survival
rates after 1, 2, and 3hr of hypoxia were 60 %, 30 % and 20 %, respectively. In the presence of calpain inhibitors I, II, and III, or calcium chelate compounds, survival rates were largely recovered while caspase inhibitor ICE 1 was slightly effective. Combinational use of these compounds showed synergetic protective effects. Contribution rates of necrosis and apoptosis were analyzed by acridine orange/ethidium bromide methods. Percentages of necrosis at 6hr, 12hr, 24hr were 62 %, 65 % and 68 % and those of apoptosis were 5 〜8 %, 7 〜 12 % and 7.6 〜 15%.
3. Intranuclear substrates of μ-calpain. Several candidates of μ-calpain substrates were examined. NF-κBp65, Pit-1, Oct-1, GHF-1 and CREB were found to be cleaved by μ-calpain.
Data suggested that high sensitivity of neurons to hypoxia is due to the rapid activation of pro μ-calpain in the nuclei of neurons. Through this activation, intranuclear physiologically important proteins should be digested and result in the neuronal death through mostly necrosis and partly apoptosis. Neuronal death is efficiently protected with calpain inhibitors I, II and III and calcium chelate compounds. It is noted that combinational use of these compounds is synergetic suggesting that appropriate combination would be useful for the therapeutic use of ischemic damage of the brain. Less