Mitsuru Sanada

Diabetic neuropathy and nerve regeneration.

Diabetic neuropathy is the most common peripheral neuropathy in western countries. Although every effort has been made to clarify the pathogenic mechanism of diabetic neuropathy, thereby devising its ideal therapeutic drugs, neither convinced hypotheses nor unequivocally effective drugs have been established. In view of the pathologic basis for the treatment of diabetic neuropathy, it is important to enhance nerve regeneration as well as prevent nerve degeneration. Nerve regeneration or sprouting in diabetes may occur not only in the nerve trunk but also in the dermis and around dorsal root ganglion neurons, thereby being implicated in the generation of pain sensation. Thus, inadequate nerve regeneration unequivocally contributes to the pathophysiologic mechanism of diabetic neuropathy. In this context, the research on nerve regeneration in diabetes should be more accelerated. Indeed, nerve regenerative capacity has been shown to be decreased in diabetic patients as well as in diabetic animals. Disturbed nerve regeneration in diabetes has been ascribed at least in part to all or some of decreased levels of neurotrophic factors, decreased expression of their receptors, altered cellular signal pathways and/or abnormal expression of cell adhesion molecules, although the mechanisms of their changes remain almost unclear. In addition to their steady-state changes in diabetes, nerve injury induces injury-specific changes in individual neurotrophic factors, their receptors and their intracellular signal pathways, which are closely linked with altered neuronal function, varying from neuronal survival and neurite extension/nerve regeneration to apoptosis. Although it is essential to clarify those changes for understanding the mechanism of disturbed nerve regeneration in diabetes, very few data are now available. Rationally accepted replacement therapy with neurotrophic factors has not provided any success in treating diabetic neuropathy. Aside from adverse effects of those factors, more rigorous consideration for their delivery system may be needed for any possible success. Although conventional therapeutic drugs like aldose reductase (AR) inhibitors and vasodilators have been shown to enhance nerve regeneration, their efficacy should be strictly evaluated with respect to nerve regenerative capacity. For this purpose, especially clinically, skin biopsy, by which cutaneous nerve pathology including nerve regeneration can be morphometrically evaluated, might be a safe and useful examination.

Increase in intracellular Ca2+ and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons

We examined the effects of the activation of metabotropic P2Y receptors on the intracellular Ca2+ concentration and the release of neuropeptide calcitonin gene-related peptide (CGRP) in isolated adult rat dorsal root ganglion neurons. In small-sized dorsal root ganglion neurons (soma diameter<30 μm) loaded with fura-2, a bath application of ATP (100 μM) evoked an increase in intracellular Ca2+ concentration, while the removal of extracellular Ca2+ partly depressed the response to ATP, thus suggesting that the ATP-induced increase in intracellular Ca2+ concentration is due to both the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. Bath application of uridine 5′-triphosphate (UTP; 100 μM) also caused an increase in intracellular Ca2+ concentration in small-sized dorsal root ganglion neurons and the P2 receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 10 μM) virtually abolished the response, indicating that the intracellular Ca2+ elevation in response to UTP is mediated through metabotropic P2Y receptors. This intracellular Ca2+ increase was abolished by pretreating the neurons with thapsigargin (100 nM), suggesting that the UTP-induced increase in intracellular Ca2+ is primarily due to the release of Ca2+ from endoplasmic reticulum Ca2+ stores. An enzyme-linked immunosorbent assay showed that an application of UTP (100 μM) significantly stimulated the release of CGRP and that suramin (100 μM) totally abolished the response, suggesting that P2Y receptor-mediated increase in intracellular Ca2+ is accompanied by CGRP release from dorsal root ganglion neurons. These results suggest that metabotropic P2Y receptors contribute to extracellular ATP-induced increase in intracellular Ca2+ concentration and subsequent release of neuropeptide CGRP in rat dorsal root ganglion neurons.

Inactivation of TNF-α ameliorates diabetic neuropathy in mice

Tumor necrosis factor (TNF)-α is a potent proinflammatory cytokine involved in the pathogenesis of diabetic neuropathy. We inactivated TNF-α to determine if it is a valid therapeutic target for the treatment of diabetic neuropathy. We effected the inactivation in diabetic neuropathy using two approaches: by genetic inactivation of TNF-α (TNF-α(-/-) mice) or by neutralization of TNF-α protein using the monoclonal antibody infliximab. We induced diabetes using streptozotocin in wild-type and TNF-α(-/-) mice. We measured serum TNF-α concentration and the level of TNF-α mRNA in the dorsal root ganglion (DRG) and evaluated nerve function by a combination of motor (MNCV) and sensory (SNCV) nerve conduction velocities and tail flick test, as well as cytological analysis of intraepidermal nerve fiber density (IENFD) and immunostaining of DRG for NF-κB p65 serine-276 phosphorylated and cleaved caspase-3. Compared with nondiabetic mice, TNF-α(+/+) diabetic mice displayed significant impairments of MNCV, SNCV, tail flick test, and IENFD as well as increased expression of NF-κB p65 and cleaved caspase-3 in their DRG. In contrast, although nondiabetic TNF-α(-/-) mice showed mild abnormalities of IENFD under basal conditions, diabetic TNF-α(-/-) mice showed no evidence of abnormal nerve function tests compared with nondiabetic mice. A single injection of infliximab in diabetic TNF-α(+/+) mice led to suppression of the increased serum TNF-α and amelioration of the electrophysiological and biochemical deficits for at least 4 wk. Moreover, the increased TNF-α mRNA expression in diabetic DRG was also attenuated by infliximab, suggesting infliximabs effects may involve the local suppression of TNF-α. Infliximab, an agent currently in clinical use, is effective in targeting TNF-α action and expression and amelioration of diabetic neuropathy in mice.

Properties of the Na+/K+ pump current in small neurons from adult rat dorsal root ganglia

The present investigation was undertaken to characterize the Na+/K+ pump current in small (25 μm in soma diameter) dorsal root ganglion (DRG) neurons isolated from lumbar L4‐6 segments of adult rats. The Na+/K+ pump current was identified as an ouabain‐sensitive current during square voltage steps to membrane potentials between +40 and −120 mV, using the whole‐cell patch‐clamp technique in which Ca2+ and K+ channel currents and Na+/Ca2+ exchange currents were minimized. The Na+/K+ pump current was practically time‐independent over the entire voltage range examined and exhibited a voltage‐dependence; its current – voltage (I–V) relationship displayed a positive slope at potentials between −120 and 0 mV but nearly plateau levels at positive membrane potentials. The concentration‐dependent block of Na+/K+ pump current (activated by 30 mM pipette Na+) by ouabain at concentrations between 0.1 μM and 5 mM was biphasic and was well described using a two‐binding site model with dissociation constants for high‐ and low‐affinity binding sites of 0.20 and 140.1 μM, respectively. The relative amplitude of the Na+/K+ pump current produced by low‐ and high‐affinity sites (probably α1β1 and α3β1 isozymes, respectively) was estimated to be 13 : 1 in the presence of 30 mM Na+ in the pipette solution. Additionally, the activation of Na+/K+ pump current by pipette Na+ at concentrations ranging from 5 to 100 mM also exhibited a biphasic concentration dependence which can be reasonably well fitted by assuming the existence of two isozymes having high and low affinities for Na+ (6.7 and 67.6 mM, respectively). Thus, the present investigation provides functional evidence to suggest that the Na+/K+ ATPase comprises two functionally distinct isozymes as expected for α1β1 and α3β1 in rat small DRG neurons.

Hereditary motor and sensory neuropathy (proximal dominant form, HMSN-P) among Brazilians of Japanese ancestry.

Hereditary motor and sensory neuropathy (proximal dominant form, HMSN-P) has been reported exclusively from Okinawa Prefecture in Japan. We herein report three brothers with HMSN-P who are among Brazilians of Japanese ancestry. They showed the typical clinical manifestations and were compatible with HMSN-P. Okinawa Prefecture has been a site of emigration to other countries, mainly in South America, since 1908. Although this is the first reported familial case of HMSN-P occurring outside Japan, it is estimated that there are 19 or 20 individuals with HMSN-P among these emigrants. Since HMSN-P might be misdiagnosed as familial amyotrophic lateral sclerosis or spinal muscular atrophy, neurologists in countries where individuals of Okinawan extraction reside should be aware of this hereditary neuropathy. HMSN-P should no longer be regarded as an endemic condition limited to Okinawa.

Inhibitory action of protein kinase Cβ inhibitor on tetrodotoxin-resistant Na+ current in small dorsal root ganglion neurons in diabetic rats

Experimental evidence has been presented to suggest that protein kinase Cbeta isoform-selective inhibitor LY333531 is effective at alleviating diabetic hyperalgesia. In the present study, we isolated small (< or =25 microm in soma diameter) dorsal root ganglion (DRG) neurons from control and streptozocin (STZ)-induced diabetic rats, and examined the acute action of LY333531 (1-1000 nM) on the tetrodotoxin-resistant Na(+) current (TTX-R I(Na)), which plays an essential role in transmitting nociceptive impulses, using the whole-cell patch-clamp method. TTX-R I(Na) in diabetic DRG neurons was enhanced in amplitude (71.5+/-3.6pA/pF, n=10 versus 41.2+/-3.3pA/pF, n=8) and was activated at more negative potentials (V(1/2), -15.1+/-1.3 mV versus -9.6+/-1.4 mV), compared with that in control neurons. Bath application of LY333531 acutely inhibited TTX-R I(Na) in both control and diabetic DRG neurons, and the degree of inhibition by the drug at concentrations of 1, 10 and 100 nM was significantly greater in diabetic DRG neurons than in control DRG neurons. Thus, TTX-R I(Na), which is upregulated in the diabetic state, is likely to be more potently inhibited by submicromolar concentrations of LY333531. These results suggest that an acute inhibition of TTX-R I(Na) by LY333531 attenuates the exaggerated excitability of DRG neurons in the diabetic state, which appears to be related at least partly to anti-hyperalgesic actions of the drug in diabetic neuropathy.

Presence and functional role of the rapidly activating delayed rectifier K+ current in left and right atria of adult mice

Repolarization of cardiac action potentials is regulated by several types of K(+) currents. The present study examined the presence and functional significance of rapid delayed rectifier (I(Kr)) in left and right atrial myocytes of mouse heart, using whole-cell patch-clamp method. The functional role of ultrarapid delayed rectifier (I(Kur)) in the repolarization was also examined by blocking with 4-aminopyridine (50 μM). The presence of I(Kr) was detected in left and right atrial myocytes as an E-4031 (5 μM)-sensitive current that exhibited relatively rapid activation during depolarization and half activation voltage of -17.5 and -17.4 mV for left and right atrial myocytes, respectively. The current density of I(Kr) was similar between left and right atria. The prolongation of action potential measured at 50% repolarization evoked by 4-aminopyridine was significantly larger in left than in right atrium, which appears to be consistent with the larger amplitude of I(Kur) in left atrium. On the other hand, the prolongation of action potential measured at 90% repolarization caused by E-4031 was significantly larger in right than in left atrium. The longer action potential of right atrium, which may result at least partly from smaller amplitude of I(Kur), is likely to enhance the functional significance of I(Kr) in repolarization process of right atrium, despite of similar magnitude of I(Kr) in left and right atria. Our data thus identifies I(Kr) in mouse atria and indicates the presence of functional interaction between I(Kr) and I(Kur) that potentially contributes to repolarization heterogeneity in left and right atria of mouse heart.

Clinical Phenotype and Segregation of Mitochondrial 3243A>G Mutation in 2 Pairs of Monozygotic Twins.

IMPORTANCE The regulatory factors explaining the wide spectrum of clinical phenotypes for mitochondrial 3243A>G mutation are not known. Crosstalk between nuclear genes and mitochondrial DNA might be one factor. OBSERVATIONS In this case series, we compared 2 pairs of male twins with the mitochondrial 3243 A>G mutation and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome with a female control patient. One pair of monozygotic twins presented with diabetes and deafness in their 30s, stroke-like episodes in their 40s, and cardiac events and death in their 50s. Another pair of twins presented with deafness and stroke-like episodes in their 20s. The degree of heteroplasmy of 3243A>G mutation in the various tissues and organs was similar in the first pair of twins compared with the control patient. CONCLUSIONS AND RELEVANCE The clinical phenotype and segregation of mitochondrial 3243A>G mutation was similar in monozygotic twins. The onset age and distribution of the symptoms might be regulated by nuclear genes. Our findings might help to predict the clinical course of the surviving twins and afford an opportunity for therapy before the onset of mitochondrial disease, especially for monozygotic twins caused by nuclear transfer with a small amount of nuclear-donor mitochondrial DNA.

Effect of protein kinase Cbeta inhibitor on Ca2+ homeostasis in diabetic sensory neurons.

To elucidate the direct effect of selective protein kinase C&bgr; inhibitor LY333531 on diabetic sensory neurons, we examined intracellular Ca2+ concentration in isolated rat dorsal root ganglion neurons using the fluorescent Ca2+ indicator fura-2. The duration of calcium transients induced by high (50 mM) extracellular K+ in small diabetic dorsal root ganglion neurons was significantly prolonged compared with that in control neurons. This prolonged intracellular Ca2+ concentration elevation in diabetic neurons was normalized rapidly and reversibly by LY333531 in a dose-dependent manner, and the effect of LY333531 was completely abolished by pretreating the neurons with mitochondrial calcium uniporter inhibitor, Ruthenium 360. These results suggest that LY333531 has an ameliorating effect on calcium homeostasis of diabetic sensory neurons via mitochondrial calcium buffering.

Cytosolic Ca2+ under high glucose with suppressed Na+/K+ pump activity in rat sensory neurons.

Cytosolic Ca2+ concentration ([Ca2+]i) was measured in isolated rat dorsal root ganglion (DRG) neurons using the fluorescent Ca2+ indicator fura-2. Exposure to high (50 mM) extracellular K+ evoked a robust increase in [Ca2+]i, which was almost totally abolished by concomitant presence of nisoldipine (10 μM) and &ohgr;-conotoxin GVIA (10 μM). Whereas either high (30 mM) D-glucose alone or ouabain (100 μM) alone did not appreciably affect the high K+-induced [Ca2+]i elevation, neurons pretreated with high D-glucose together with ouabain exhibited a significantly larger [Ca2+]i response to high K+ stimulation, which was almost completely inhibited by nisoldipine and &ohgr;-conotoxin GVIA. These results suggest that a combination of high glucose and suppressed Na+/K+ pump activity potentiates the [Ca2+]i elevation stimulated by activation of the voltage-gated Ca2+ channels in rat DRG neurons.