Shiro Fukuda

Postischemic Reperfusion Induces α‐Fodrin Proteolysis by m‐Calpain in the Synaptosome and Nucleus in Rat Brain

Abstract: A membrane cytoskeletal protein, fodrin, is a substrate for a Ca2+‐dependent protease, calpain. It remains unknown whether μ‐calpain or m‐calpain is involved in the proteolysis of either α‐ or β‐fodrin and in what subcellular localization during ischemia and reperfusion of the brain. To address these issues, we examined the distribution of fodrin and calpain and the activities of calpain and calpastatin (endogenous calpain inhibitor) in the same subcellular fractions. Rat forebrain was subjected to ischemia by a combination of occlusion of both carotid arteries and systemic hypotension, whereas reperfusion was induced by releasing the occlusion. Immunoblotting, activity measurement, and casein zymography did not detect the presence of μ‐calpain or a significant change of m‐calpain level after ischemia or reperfusion. However, casein zymography revealed a unique Ca2+‐dependent protease that was eluted with both 0.18 and 0.40 M NaCl from a DEAE‐cellulose column. α‐ and β‐fodrins and m‐calpain were found to be rich in the synaptosomal, nuclear, and cytosolic subfractions by immunoblotting analysis. Reperfusion (60 min) following ischemia (30 min) induced selective proteolysis of α‐fodrin, which was inhibited by a calpain inhibitor, acetylleucylleucylnorleucinal (400 µM, 1 ml, i.v.). The μ‐calpain‐specific fragment of β‐fodrin was not generated during ischemia‐reperfusion, supporting the possibility of the involvement of m‐calpain rather than μ‐calpain in the α‐fodrin proteolysis.

The effects of cyclosporin A and insulin on ischemic spinal cord injury in rabbits.

We examined the effects of cyclosporin A (CsA), a drug that inhibits mitochondrial permeability transition pore, and insulin on ischemic spinal cord damage in rabbits. We assigned rabbits to 5 groups (n = 6 in each); sham barrier-opened group (sham BO), barrier-opened group (BO), barrier-opened-CsA group (BO-CsA), barrier-opened-insulin group (BO-I), and barrier-opened-CsA-insulin group (BO-CsA-I). The blood-spinal cord barrier was opened to facilitate drug penetration by a mild injury to the lumber spinal cord on day 1. CsA (10 mg/kg per day IV) was administered on day 3 to day 5 (total 30 mg/kg). Insulin was administered 30 min before ischemia. In all groups, spinal cord ischemia was produced on day 5 by occluding the abdominal aorta for 13 min. Neurological and histopathological evaluations were performed 4 days after ischemia. In group BO-CsA, blood glucose concentrations were significantly larger compared with the other four groups, and no protection was observed. In contrast, hindlimb motor function in groups BO-I and Bo-CsA-I and histopathology in group BO-CsA-I were significantly better than in groups sham BO, BO, and BO-CsA. The results indicate that insulin protects against ischemic spinal cord injury, whereas the effect of CsA is, at best, minimal.