Mitsuhiro Makino

Sorbitol dehydrogenase overexpression potentiates glucose toxicity to cultured retinal pericytes.

The polyol pathway consists of two enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH). There is a growing body of evidence to suggest that acceleration of the polyol pathway is implicated in the pathogenesis of diabetic vascular complications. However, a functional role remains to be elucidated for SDH in the development and progression of diabetic retinopathy. In this study, cultured bovine retinal capillary pericytes were used to investigate the effects of SDH overexpression on glucose toxicity. High glucose modestly increased reactive oxygen species (ROS) generation, decreased DNA synthesis, and up-regulated vascular endothelial growth factor (VEGF) mRNA levels in cultured pericytes. SDH overexpression was found to significantly stimulate ROS generation in high glucose-exposed pericytes and subsequently potentiate the cytopathic effects of glucose. Fidarestat, a newly developed AR inhibitor, and N-acetylcysteine, an antioxidant, completely prevented these deleterious effects of SDH overexpression on pericytes. Furthermore, fidarestat administration was found to significantly prevent vascular hyperpermeability, the characteristic changes of the early phase of diabetic retinopathy, in streptozotocin-induced diabetic rats. Our present results suggest that SDH-mediated conversion of sorbitol to fructose and the resultant ROS generation may play an active role in the pathogenesis of diabetic retinopathy. Blockage of sorbitol formation by fidarestat could be a promising therapeutic strategy for the treatment of early phase of diabetic retinopathy.

Enhancement of immobility in mouse forced swimming test by treatment with human interferon

We investigated the depression induced by human interferons using the forced swimming test in mice. Intravenous (i.v.) administration of interferon-alpha s (natural interferon-alpha, recombinant interferon-alpha-2a and recombinant interferon-alpha-2b, 600-60000 IU/kg) increased the immobility time in the forced swimming test in a dose-dependent manner, but natural interferon-beta and recombinant interferon-gamma-1a did not affect the immobility time. The increase in the immobility time induced by recombinant interferon-alpha-2b peaked at 15 min after dosing. Administration of recombinant interferon-alpha-2b (6000 IU/kg, i.v.) once daily for 7 consecutive days increased the immobility time, but natural interferon-beta and recombinant interferon-gamma-la did not. Recombinant interferon-alpha-2b in combination with the anti-depressants imipramine (10 mg/kg, i.p.) and mianserin (20 mg/kg, i.p.) did not increase the immobility time. These results suggest that interferon-alpha has a greater potential for inducing depression than interferon-beta and -gamma, and that anti-depressants are effective against interferon-alpha-induced depression.

Involvement of central opioid systems in human interferon-α induced immobility in the mouse forced swimming test

We investigated the mechanism by which human interferon‐α (IFN‐α) increases the immobility time in a forced swimming test, an animal model of depression. Central administration of IFN‐α (0.05–50 IU per mouse, i.cist.) increased the immobility time in the forced swimming test in mice in a dose‐dependent manner. Neither IFN‐β nor ‐γ possessed any effect under the same experimental conditions. Pre‐treatment with an opioid receptor antagonist, naloxone (1 mg kg−1, s.c.) inhibited the prolonged immobility time induced by IFN‐α (60 KIU kg−1, i.v. or 50 IU per mouse. i.cist.). Peripheral administration of naloxone methiodide (1 mg kg−1, s.c.), which does not pass the blood–brain barrier, failed to block the effect of IFN‐α, while intracisternal administration of naloxone methiodide (1 nmol per mouse) completely blocked. The effect of IFN‐α was inhibited by a μ1‐specific opioid receptor antagonist, naloxonazine (35 mg kg−1, s.c.) and a μ1/μ2 receptor antagonist, β‐FNA (40 mg kg−1, s.c.). A selective δ‐opioid receptor antagonist, naltrindole (3 mg kg−1, s.c.) and a κ‐opioid receptor antagonist, nor‐binaltorphimine (20 mg kg−1, s.c.), both failed to inhibit the increasing effect of IFN‐α. These results suggest that the activator of the central opioid receptors of the μ1‐subtype might be related to the prolonged immobility time of IFN‐α, but δ and κ‐opioid receptors most likely are not involved.

Continuous Inhibition of Excessive Polyol Pathway Flux in Peripheral Nerves by Aldose Reductase Inhibitor Fidarestat Leads to Improvement of Diabetic Neuropathy

We investigated the effects of three aldose reductase (AR) inhibitors, fidarestat, epalrestat and zenarestat, on the slowing of sensory nerve conduction velocity (SNCV), motor nerve conduction velocity (MNCV), and minimal F-wave latency prolongation in streptozotocin (STZ)-induced diabetic rats. Two weeks after STZ injection, SNCV and MNCV in the diabetic rats were significantly slower than in normal rats. Fidarestat (0.25-2 mg/kg/day), epalrestat (48 to 96 mg/kg/day) or zenarestat (10-40 mg/kg/day) was administered orally for the following 2 weeks, and SNCV, MNCV and F-wave latency were measured 3 h after final administration. Significant prolongation of minimal F-wave latency, as well as slowing of SNCV and MNCV, was found in the untreated diabetic rats 4 weeks after STZ injection. At a dose of 0.5 mg/kg/day or more fidarestat showed significant effects on these nervous dysfunctions, effects that were more potent than those shown by the other inhibitors. Furthermore, following the 2-week administration of fidarestat (1 mg/kg/day), epalrestat (48 mg/kg/day) or zenarestat (20 mg/kg/day), which began 2 weeks after STZ injection, sorbitol content in the sciatic nerve, produced by AR, a rate-limiting enzyme in the polyol pathway, was determined at 3, 8, 12, and 24 h after final administration. At each point in time, sorbitol content in the untreated diabetic rats was much higher than that in the normal control rats. Fidarestat suppressed sorbitol accumulation remarkably and continuously until 24 h after administration. On the other hand, the inhibitory effect by zenarestat declined in a time-dependent manner, and epalrestat did not decrease sorbitol content. Therefore, these results suggest that continuous inhibition of increased polyol pathway flux can improve diabetic neuropathy more potently.

Human interferon-α induces immobility in the mouse forced swimming test: involvement of the opioid system

In a previous study, we indicated that human interferon (IFN)-alpha (IFN-alpha, 6 x 10(4) IU/kg, i.v.), but not human IFN-beta or -gamma, prolonged the immobility time of the forced swimming test in mice. In this study, we investigated the mechanism of the effect of human IFN-alpha. None of the mouse IFNs tested (IFN-alpha/beta, IFN-beta, and IFN-gamma, 3 x 10(5) U/kg, i.v.) changed the immobility time or the spontaneous locomotor activity in mice. Indomethacin (10 mg/kg, s.c.), a cyclooxygenase inhibitor, did not affect the increase in the immobility time induced by human IFN-alpha (6 x 10(4) IU/kg, i.v.). However, naloxone (1 mg/kg, s.c.), an opioid receptor antagonist, blocked the increasing caused by human IFN-alpha in the forced swimming test. These results suggest that the increase in the immobility time caused by human IFN-alpha in the forced swimming test might be mediated through opioid receptors, but not mouse IFN receptors.

Degradation of PEP-19, a calmodulin-binding protein, by calpain is implicated in neuronal cell death induced by intracellular Ca2+ overload.

Excessive elevation of intracellular Ca2+ levels and, subsequently, hyperactivation of Ca2+/calmodulin-dependent processes might play an important role in the pathologic events following cerebral ischemia. PEP-19 is a neuronally expressed polypeptide that acts as an endogenous negative regulator of calmodulin by inhibiting the association of calmodulin with enzymes and other proteins. The aims of the present study were to investigate the effect of PEP-19 overexpression on cell death triggered by Ca2+ overload and how the polypeptide levels are affected by glutamate-induced excitotoxicity and cerebral ischemia. Expression of PEP-19 in HEK293T cells suppressed calmodulin-dependent signaling and protected against cell death elicited by Ca2+ ionophore. Likewise, primary cortical neurons overexpressing PEP-19 became resistant to glutamate-induced cell death. In immunoprecipitation assay, wild type PEP-19 associated with calmodulin, whereas mutated PEP-19, which contains mutations within the calmodulin binding site of PEP-19, failed to associate with calmodulin. We found that the mutation abrogates both the ability to suppress calmodulin-dependent signaling and to protect cells from death. Additionally, the endogenous PEP-19 levels in neurons were significantly reduced following glutamate exposure, this reduction precedes neuronal cell death and can be blocked by treatment with calpain inhibitors. These data suggest that PEP-19 is a substrate for calpain, and that the decreased PEP-19 levels result from its degradation by calpain. A similar reduction of PEP-19 also occurred in the hippocampus of gerbils subjected to transient global ischemia. In contrast to the reduction in PEP-19, no changes in calmodulin occurred following excitotoxicity, suggesting the loss of negative regulation of calmodulin by PEP-19. Taken together, these results provide evidence that PEP-19 overexpression enhances resistance to Ca2+-mediated cytotoxicity, which might be mediated through calmodulin inhibition, and also raises the possibility that PEP-19 degradation by calpain might produce an aberrant activation of calmodulin functions, which in turn causes neuronal cell death.

Serial changes of sensory nerve conduction velocity and minimal F-wave latency in streptozotocin-induced diabetic rats

We studied the serial changes of sensory nerve conduction velocity (SNCV) in the caudal nerve of streptozotocin (STZ)-induced diabetic rats using a new technical method. Minimal F-wave latency was also studied by stimulating the tibial nerve. The SNCV in the diabetic rats was slower than that in the normal rats 2 weeks after STZ injection, and minimal F-wave latency was prolonged compared to normal rats 4 weeks after STZ injection. Treatment of the diabetic rats with insulin for 14 days inhibited SNCV slowing and minimal F-wave latency prolongation. This new method to measure SNCV is useful for various studies, and improvement of diabetic neuropathy with insulin treatment is indicated by recovery from SNCV slowing and minimal F-wave latency prolongation.

Anticonvulsant Properties of the Novel Nootropic Agent Nefiracetam in Seizure Models of Mice and Rats

Summary:  Purpose: Nefiracetam (NEF) is a novel pyrrolidone‐type nootropic agent, and it has been reported to possess various pharmacologic effects as well as cognition‐enhancing effects. The present study focused on the anticonvulsant effect of NEF and its potential for antiepileptic therapy.

Anticonvulsant and neuroprotective effects of the novel nootropic agent nefiracetam on kainic acid-induced seizures in rats

Nefiracetam is a novel pyrrolidone-type nootropic agent, and it has been reported to possess a potential for antiepileptic therapy as well as cognition-enhancing effects. We investigated the anticonvulsant and neuroprotective effects of nefiracetam in kainic acid-induced seizures of rats, compared with levetiracetam and standard antiepileptic drugs. Subcutaneous injection of kainic acid (10 mg/kg) induced typical behavioral seizures such as wet dog shakes and limbic seizures and histopathological changes in the hippocampus (degeneration and loss of pyramidal cells in CA1 to CA4 areas). Nefiracetam (25, 50 and 100 mg/kg po) had no effect on the behavioral seizures and dose-dependently inhibited the hippocampal damage. In contrast, levetiracetam, a pyrrolidone-type antiepileptic drug, inhibited neither. Valproic acid and ethosuximide prevented the hippocampal damage without attenuating the behavioral seizures as nefiracetam. Zonisamide and phenytoin did not inhibit the behavioral seizures, while zonisamide enhanced the hippocampal damage and phenytoin increased the lethality rate. Carbamazepine inhibited the behavioral seizures at 50 mg/kg and enhanced that at 100 mg/kg, and it completely inhibited the hippocampal damage at both doses. We have previously reported that anticonvulsant spectrum of nefiracetam paralleled that of zonisamide, phenytoin or carbamazepine in standard screening models. However, the pharmacological profile of nefiracetam was closer to valproic acid or ethosuximide than that of zonisamide, phenytoin or carbamazepine in this study. These results suggest that anticonvulsant spectrum and mechanism of nefiracetam are distinct from those of standard antiepileptic drugs, and nefiracetam possesses a neuroprotective effect that is unrelated to seizure inhibition.

Involvement of calmodulin in neuronal cell death

A large body of evidence indicates that disturbances of Ca(2+) homeostasis may be a causative factor in the neurotoxicity following cerebral ischemia. However, the mechanisms by which Ca(2+) overload leads to neuronal cell death have not been fully elucidated. Calmodulin, a major intracellular Ca(2+)-binding protein found mainly in the central nervous system, mediates many physiological functions in response to changes in the intracellular Ca(2+) concentration, whereas Ca(2+) overload in neurons after excitotoxic insult may induce excessive activation of calmodulin signaling pathways, leading to neuronal cell death. To determine the role of calmodulin in the induction of neuronal cell death, we generated primary rat cortical neurons that express a mutant calmodulin with a defect in Ca(2+)-binding affinity. Neurons expressing the mutant had low responses of calmodulin-dependent signaling to membrane depolarization by high KCl and became resistant to glutamate-triggered excitotoxic neuronal cell death compared with the vector or wild-type calmodulin-transfected cells, indicating that blocking calmodulin function is protective against excitotoxic insult. These results suggest that calmodulin plays a crucial role in the processes of Ca(2+)-induced neuronal cell death and the possibility that the blockage of calmodulin attenuates brain injury after cerebral ischemia.