Yoshihide Sorimachi

Reperfusion of Rat Heart After Brief Ischemia Induces Proteolysis of Calspectin (Nonerythroid Spectrin or Fodrin) by Calpain

Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to alpha- and beta-subunits of brain calspectin (nonerythroid spectrin or fodrin), respectively. In the subcellular fractions of the myocardium, alpha-calspectin was found in the 600g, 10,000g, and 100,000g pellets, whereas beta-calspectin was localized to the 10,000g pellet. On the basis of the Na+,K(+)-ATPase activity and the contents of a gap junction protein, the sarcolemma was distributed to the 10,000g and 100,000g pellets, and the intercalated disks were enriched in the 10,000g pellet. Both alpha- and beta-calspectin were proteolyzed by calpain in vitro. The two subunits were also proteolyzed in vivo, when the rat hearts underwent 10 to 60 minutes of global ischemia followed by 30 minutes of reperfusion. The reperfusion following the ischemia induced the proteolysis of alpha-calspectin in the 10,000g and 100,000g pellets, producing the 150-kD fragment. A synthetic calpain inhibitor, calpain inhibitor-1, suppressed the degradation of calspectin in vivo, which indicates that calpain is responsible for the reperfusion-induced proteolysis of calspectin. The inhibitor also improved myocardial stunning. Immunohistochemical study revealed that the proteolysis of alpha-calspectin occurs at the intercalated disks and the sarcolemma after postischemic reperfusion, in accord with the biochemical data. These results suggest that degradation of calspectin partly accounts for the contractile failure of the myocardium after postischemic reperfusion by disrupting the membrane skeleton and the intercalated disks.

A possible role for BDNF, NT-4 and TrkB in the spinal cord and muscle of rat subjected to mechanical overload, bupivacaine injection and axotomy.

Neurotrophins play a crucial role in the regulation of survival and the maintenance of specific functions for various populations of neurons. Neurotrophin-4 (NT-4) is most abundant in skeletal muscle, and is thought to promote sciatic nerve sprouting, inhibit agrin-induced acetylcholine receptor (AChR) clustering, evoke postsynaptic potentiation and induce mitochondrial proliferation. Using Western blot analysis, immunoprecipitation and immunohistochemistry, we investigated the distribution of NT-4 in slow- and fast-type muscles. We also tested the adaptive response of this protein in the mechanically overloaded muscle, in the regenerating muscle following bupivacaine injection and in the denervated muscle. Additionally, we investigated whether TrkB phosphorylation in the spinal cord and in the sciatic nerve occurs through the interaction with BDNF or NT-4 when the innervating muscle is damaged. Markedly more NT-4 was expressed in fast-type muscles compared with the slow types. TrkB protein was more frequently observed around the edge of myofibers (neuromuscular junction) of the soleus muscle compared with the gastrocnemius muscle. TrkB tyrosine phosphorylation occurred in the spinal cord but not in the sciatic nerve 24 h after bupivacaine injection of the innervating muscle. At the same time, the amount of TrkB co-precipitating with BDNF was markedly increased in the spinal cord. A rapid activation of TrkB (1-8 h) was also observed in the spinal cord after axotomy,while the amount of TrkB co-precipitating with NT-4 was markedly lower after axotomy. These results indicate that NT-4 is preferentially distributed in fast-type muscles. Furthermore, by interacting with BDNF and NT-4, the TrkB in the spinal cord may be important for the survival of motoneurons and outgrowth of injured peripheral axons following muscle damage.

Involvement of calpain in postmortem proteolysis in the rat brain

Calpain, a Ca(2+)-dependent neutral protease was examined to investigate its involvement in postmortem proteolysis in the rat brain. Western blotting analysis showed that the 240 kDa alpha-subunit of fodrin, a well-known substrate for calpain, was degraded to generate 150 kDa and 145 kDa fragments in the postmortem interval (0-24 h) at 25 +/- 3 degrees C. Postmortem proteolysis was dependent on ambient temperature. In in vitro experiments, the 150 kDa and 145 kDa fragments appeared in the homogenate with addition of Ca2+ (1 microM-1 mM) or in the microsomal fraction by incubation with purified calpain. Both calpain inhibitor-1 and leupeptin suppressed in vitro proteolysis. During the initial 0-24 h postmortem, the activity of m-calpain in the brain remained unaltered, while that of its endogenous inhibitor, calpastatin, decreased with the postmortem interval. These results indicate that calpain is involved in fodrin proteolysis in the postmortem rat brain. The ratio of the amount of the 150 kDa proteolytic product to that of the 240 kDa fodrin alpha-subunit was correlated significantly with the postmortem interval (0-16 h; r = 0.745).

Rat and human membrane dipeptidase: tissue distribution and developmental changes.

Distribution and developmental changes in membrane dipeptidase activity were examined in rat and human tissues. The activity to hydrolyze glycyl-D-alanine in rat and human tissues was completely or almost completely inhibited by 5 mM cilastatin, suggesting that the activity was due to membrane dipeptidase and that the contribution of leucine aminopeptidase to the activity was minor. In 8-week-old rats, the activity was high in lung, kidney, pancreas and testis, and in each pooled sample of ileal mucosa, duodenal mucosa, jejunal mucosa and adrenal mucosa. A low activity was found in spleen, liver, serum and heart. The activity in lung, kidney, adrenal and intestinal mucosa increased up to the age of 5 or 8 weeks, while that in pancreas, testis and spleen reached a maximal level at around 3 weeks and declined thereafter. The distribution profile of the enzyme in postmortem tissues of adult humans was similar to that in rat, except for an extremely low activity in lung. The enzyme was also found in serum and urine from healthy volunteers. In urine, the activity was significantly correlated to the creatinine content. No clear dependence of the activity on gender or age was observed in urine and serum.

Effects of age and ethanol on dopamine and serotonin release in the rat nucleus accumbens

Neural functions in the nucleus accumbens (ACC) play an important role in alcohol drinking behavior. In the present study, we observed the effects of age and ethanol (EtOH) on dopamine (DA) and serotonin (5-HT) release in the ACC of freely moving 4-, 10-, and 16-month-old rats using brain microdialysis techniques. After co-perfusion with 200 mM ethanol, ACC DA, and 5-HT release were decreased significantly in 16-month-old rats compared to those at 4 months old. ACC DA and 5-HT neurons of aged rats were less sensitive to ethanol. On the other hand, both basal extracellular DA and 5-HT release in the ACC were significantly higher in 16-month-old than in 4-month-old rats. Therefore, aging results in opposite changes in basal and alcohol-induced DA and 5-HT release in the ACC.

Increase of Cardiotrophin-1 immunoreactivity in regenerating and overloaded but not denervated muscles of rats.

The original report by Pennica et al. on Cardiotrophin‐1 (CT‐1) states that it markedly stimulates hypertrophy in cardiac myocytes both in vitro and in vivo and is predominantly expressed in the early mouse embryonic heart tube. CT‐1 is a member of the interleukin‐6 superfamily and past studies have shown that it exerts trophic effects on neurons, glial cells and their precursors, and is expressed during myogenesis. Thus CT‐1 is associated with physical and pathological changes in skeletal muscle. In this study, we examined whether CT‐1 is expressed in mechanically overloaded, regenerating, and denervated muscles of rats using immunohistochemistry. In the overloaded plantaris muscles at 1 and 3 days postsurgery, CT‐1 immunoreactivity was detected in the mononuclear cells that had infiltrated the extracellular space. CT‐1 immunoreactivity was also observed in the mononuclear cells invading the extracellular space at 2, 4, and 6 days after a bupivacaine injection and in degenerative and necrotic muscle fibers at 2 days postinjection. In the denervated muscles, the CT‐1 immunoreactivity did not change in intensity during the entire period of the denervation (2, 7, and 14 days postsurgery). The cells invading extracellular space and in necrotic muscle fibers possessing CT‐1 immunoreactivity might be muscle precursor cells (satellite cells) or migrating macrophages undergoing phagocytosis. Using double‐immunostainings for anti‐CT‐1/antic‐met, anti‐CT‐1/ anti‐M‐cadherin, and anti‐CT‐1/anti‐ED1, we found that satellite cells and macrophages exhibited CT‐1 immunoreactivity in the damaged muscles after bupivacaine injection. We therefore believe that CT‐1 plays a key role in regeneration and hypertrophy in the skeletal muscle of rats.

Risk of death due to alcohol-impaired driving in Japan

It is noted that after WWII, the number of motor vehicles increased rapidly in Japan. This increase was accompanied by a rise in the number of road traffic accidents (RTAs) and of alcohol-related traffic violations. In 1970, the Government of Japan introduced a law setting the maximum breath alcohol concentration at 0725 mg/L. The law was revised in 1978 to increase penalties for drivers violating this law. The authors briefly discuss findings of their determinations of the relative risks of fatal RTAs due to alcohol-impaired driving in Japan.

Proteolysis of ankyrin and Na+/K+-ATPase in postmortem rat brain: is calpain involved?

Ankyrin links the fodrin-based cytoskeleton to membrane proteins such as Na+/K(+)-ATPase, thereby maintaining cellular integrity. Immunoblotting by antibody raised against erythrocyte ankyrin demonstrated the proteolysis of ankyrin, which was highly correlated with postmortem interval (0-24 h). Proteolysis in the postmortem brain generated the 160-kDa fragment with an identical size as the fragment formed after in vitro proteolysis by calpain. Although microM Ca2+ induced the proteolysis in the homogenate, the presence of mu-calpain was not demonstrated by immunoblotting using the antibody that reacts with large subunits both of mu- and m-calpains. Na+/K(+)-ATPase was also proteolyzed in the postmortem brain.

Relationships between the deinstitutionalization of healthcare for patients with mental disorder, substance abuse, and isolated death.

Isolated death (ID) (i.e., dying alone without anyone noticing for several days) has been suggested to be related to social isolation, mental disorder, and alcohol and/or drug abuse. A major transfer of patients with a mental disorder and/or alcohol and/or drug abuse from institutionalized care to treatment as outpatients has been enacted in Sweden during the past decade. On the basis of the assumption that such deinstitutionalization is likely to result in increased social isolation, our working hypothesis was that the incidence of ID among patients belonging to these categories has increased in Sweden. The present study involved all deaths subjected to a medicolegal examination in Stockholm County (with a population of approximately 1.9 million people) during the period 1992-2000. The pattern of ID (defined as cases involving a postmortem delay between death and discovery of at least 1 week), as well as the incidence of fatalities subjected to medicolegal examination with a record of mental disorder and/or alcohol and/or drug abuse was evaluated. Throughout this period, the proportion of the deceased with a record of a mental disorder was high among all the cases examined and higher still among the cases of ID, especially among those younger than 65 years of age. There was a rather limited increase in the incidence of ID and a much more pronounced increase in the number of former psychiatric patients whose deaths were subjected to medicolegal examination, but did not satisfy the criteria for ID. A record of alcohol and/or drug abuse was more common than a diagnosis of mental disorder among both the males and females who died at an age of less than 65, with a clear difference between the cases of ID and non-ID in the case of men. There was no significant increase in incidence over the course of this study. Thus, this study reveals a slight increase in the number of IDs and a more pronounced increase in the number of medicolegal examination of non-IDs of individuals with a record of a mental disorder.

The reciprocal change of neurotrophin-4 and glial cell line-derived neurotrophic factor protein in the muscles, spinal cord and cerebellum of the dy mouse.

Abstract. Laminin α2 (merosin)-deficient congenital muscular dystrophy (CMD) patients show progressive muscle fiber necrosis and ineffective muscle regeneration, probably due to a lower formation of multinucleated myotubes due to an adhesion defect of myoblasts to each other. Some recent studies found that CMD patients have a white matter disorder and cerebellum atrophy. In the spinal cord of dy mice, a model of CMD, inducible nitric oxide synthase (iNOS) was markedly expressed. Using Western blotting and immunohistochemical analyses, we investigated the levels of neurotrophin-4 (NT-4), brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) in the central nervous system and skeletal muscles of dy mice. In the dy mice, the microtubule-associated protein-2 (MAP-2) protein level was markedly decreased in the Purkinje and granule cells of the cerebellum, and in lumbar motoneurons of the spinal cord. The motoneurons and axons of dy mice possessed lower expressions of phosphorylated tau. The amount of NT-4 was markedly lower in the cerebellum, spinal cord and hindlimb muscles of dy mice. In dy mice, GDNF was markedly enhanced in the Purkinje and granule cells of the cerebellum, in many lumbar motoneurons, and in the regenerating atrophied fibers. The CNTF protein level did not differ in the hindlimb muscles between the normal and dy mice. Therefore, GDNF could act to inhibit the death of Purkinje and granular neurons, and motoneurons, and to promote the remodeling of the neuromuscular junction of atrophied muscle fibers of dy mice. Furthermore, dy mice include neurogenic abnormalities in the cerebellum and spinal cord along with myogenic disorder of muscle fibers.