Hiroshi Kitasato

Tetrodotoxin-resistant sodium channels of dorsal root ganglion neurons are readily activated in diabetic rats.

To clarify the mechanism of hyperalgesia in diabetic neuropathy, we investigated the effects of streptozocin-induced hyperglycemia on tetrodotoxin-resistant Na+ channel activity of dorsal root ganglion neurons. Experiments were performed on enzymatically isolated neurons of dorsal root ganglia dissected from streptozocin-induced diabetic and their age-matched control rats. Membrane currents were recorded using the whole-cell patch-clamp technique. Mean current density of tetrodotoxin-resistant Na+ channels was significantly larger in neurons prepared from diabetic rats than in control neurons. Tetrodotoxin-resistant Na+ channels were activated at more negative potentials in diabetic than in control neurons. Curves representing the steady-state inactivation and the peak Na+ conductance as a function of membrane potential shifted to the negative side. The changes in gating property of the Na+ channel were observed six weeks after the injection of streptozocin, and still after eight months, indicating that tetrodotoxin-resistant Na+ channel abnormality starts to develop early and persists during the whole period of diabetes. These results suggest that neurons participating in nociception are highly excitable in diabetic animals. The present results may provide an important clue to the elucidation of hyperalgesia in diabetes.

Cyclic AMP‐Elevating Agents Prevent Oligodendroglial Excitotoxicity

Abstract: Previously, we have demonstrated that cells of the oligodendroglial lineage express non‐NMDA glutamate receptor genes and are damaged by kainate‐induced Ca2+ influx via non‐NMDA glutamate receptor channels, representing oligodendroglial excitotoxicity. We find in the present study that agents that elevate intracellular cyclic AMP prevent oligodendroglial excitotoxicity. After oligodendrocyte‐like cells, differentiated from the CG‐4 cell line established from rat oligodendrocyte type‐2 astrocyte progenitor cells, were exposed to 2 mM kainate for 24 h, cell death was evaluated by measuring activity of lactate dehydrogenase released into the culture medium. Released lactate dehydrogenase increased about threefold when exposed to 2 mM kainate. Kainate‐induced cell death was prevented by one of the following agents: adenylate cyclase activator (forskolin), cyclic AMP analogues (dibutyryl cyclic AMP and 8‐bromo‐cyclic AMP), and cyclic AMP phosphodiesterase inhibitors (3‐isobutyl‐1‐methylxanthine, pentoxifylline, propentofylline, and ibudilast). Simultaneous addition of both forskolin and phosphodiesterase inhibitors prevented the kainate‐induced cell death in an additive manner. A remarkable increase in Ca2+ influx (∼5.5‐fold) also was induced by kainate. The cyclic AMP‐elevating agents caused a partial suppression of the kainate‐induced increase in Ca2+ influx, leading to a less prominent response of intracellular Ca2+ concentration to kainate. The suppressing effect of forskolin on the kainate‐induced Ca2+ influx was partially reversed by H‐89, an inhibitor of cyclic AMP‐dependent protein kinase. In contrast to this, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, brought about a decrease in the kainate‐induced Ca2+ influx. We therefore concluded that cyclic AMP‐elevating agents prevented oligodendroglial excitotoxicity by cyclic AMP‐dependent protein kinase‐dependent protein phosphorylation, resulting in decreased kainate‐induced Ca2+ influx.

Insulin and noradrenaline independently stimulate the translocation of glucose transporters from intracellular stores to the plasma membrane in mouse brown adipocytes

The mechanism of the effect of noradrenaline on the transport of 3‐O‐methyl‐d‐[14C]glucose ([14C]‐MG) was studied in mouse brown adipocytes. When cells were exposed to low concentrations (< 10−8 M) of insulin, the [14C]‐MG uptake by cells was enhanced by noradrenaline additively. The action of noradrenaline was mimicked by isoproterenol, and was completely blocked by propranolol. Exposing cells to noradrenaline induced both an increase in the transport activity of plasma membrane fractions and a decrease in that of microsomal fractions similar to insulin exposure, indicating that noradrenaline also induces the translocation of glucose transporters to the plasma membrane. The ratio of an increase in the transport activity of plasma membrane fraction to a decrease in the activity of the microsomal fraction was lower in cells exposed to noradrenaline than in cells exposed to insulin. This quantitative disagreement suggests that there are at least two different modes involved in the regulation of the translocation of glucose transporters in mouse brown adipocytes.

Stimulation of Na,K-ATPase activity of frog skeletal muscle by insulin

Na,K-ATPase activity of a plasma membrane fraction obtained from frog skeletal muscles was increased approximately two-fold by exposing muscles to insulin, whereas the addition of insulin to a membrane preparation suspension has no effect on Na,K-ATPase activity. The effect of insulin on Na,K-ATPase activity of whole muscles was specific to insulin and insulin derivatives that had the ability of receptor-binding and was not inhibited by actinomycin D. Insulin also induced a development of Na,K-ATPase activity in muscles whose Na,K-ATPase activity had been blocked by ouabain-pretreating. Such a insulin action was inhibited by monensin. These observations suggest that insulin stimulates the monensin-sensitive intracellular transport of membrane proteins which should be responsible for the increase in Na/K pumping activity.

Development of neuropeptide Y innervation in the liver

Hepatic neuropeptide Y (NPY) innervation was studied by immunohistochemistry in various mature vertebrates including the eel, carp, bullfrog, turtle, chicken, mouse, rat, guinea pig, dog, monkey, and human. In addition, an ontogenetic study on hepatic NPY was made in developing mice and guinea pigs. In all species examined except the eel, NPY‐like immunoreactivity was detected in nerve fibers. In the carp, bullfrog, turtle, chicken, mouse, and rat, NPY‐positive fibers were distributed around the wall of hepatic vessels and the bile duct of the Glissons sheath. The density of NPY‐positive fibers increased with evolution. However, in the guinea pig, dog, monkey, and human, numerous NPY‐positive fibers were observed not only in the Glissons sheath but also in the liver parenchyma. Positive fibers formed a dense network that surrounded the hepatocytes. The present immunoelectron microscopic study has confirmed that NPY‐positive terminals are closely apposed to hepatocytes. Ontogenically, NPY‐positive fibers were first found in the embryonic liver of 19‐day‐old mice. Positive fibers increased with age, and the highest peak was seen 1 week after birth. However, NPY‐positive nerve fibers were present abundantly in Glissons sheath and in the hepatic parenchyma of neonatal (3 and 7 days old) guinea pigs in a distribution similar to that in mature animals. This ontogenetic pattern suggests that NPY plays a certain role in the developing liver. Microsc. Res. Tech. 39:365–371, 1997.

Glucagon induces suppression of ATP-sensitive K+ channel activity through a Ca2+/calmodulin-dependent pathway in mouse pancreatic beta-cells

Abstract. Glucagon is known to increase intracellular cAMP levels and enhance glucose-induced electrical activity and insulin secretion in pancreatic β-cell perfused with Krebs-Ringer bicarbonate solution. The present experiments were aimed at evaluation of the hypothesis that changes in β-cells ATP-sensitive K+ (K(ATP)) channel activity are involved in the glucagon-induced enhancement of electrical activity. Channel activity was recorded using the cell-attached configuration of the patch-clamp technique. Addition of glucagon (2.9 × 10−7m) in the presence of 11.1 mm glucose caused closure of K(ATP) channels followed by an increase in the frequency of biphasic current transients (action currents) due to action potential generation in the cell. Three calmodulin-antagonists (W-7, chlorpromazine, and trifluoperazine) restored with similar efficacy K(ATP) channel activity in cells being exposed to glucagon. At 2.8 mm glucose, glucagon did not affect K(ATP) channel activity until Ca2+ was released from Nitr-5 by flash photolysis, at which point channel activity was transiently suppressed. Similar effects were seen when db-cAMP was used instead of glucagon.These results support the view that glucagon and other cAMP-generating agonists enhance glucose-induced β-cell electrical activity through a Ca2+/calmodulin dependent-closure of K(ATP) channels.

Phylogenetic and ontogenetic study of neuropeptide Y-containing nerves in the liver.

SummaryThe distribution of neuropeptide Y was investigated by light and electron microscopic immunohistochemistry in the liver of various vertebrates including the eel, carp, bullfrog, turtle, chicken, mouse, rat, guinea-pig, dog, monkey and human. The ontogenetic development of neuropeptide Y was also studied in the mouse liver. In all species examined except the eel, neuropeptide Y-like immunoreactivity was detected in nerve fibres. In the carp, bullfrog, turtle, chicken, mouse and rat, positive fibres were distributed around the wall of hepatic vessels and the bile duct of the Glissons sheath. The density of the positive fibres increased with evolution. On the other hand, in the guinea-pig, dog, monkey and human, numerous neuropeptide Y-positive fibres were observed not only in the Glissons sheath but also in the liver parenchyma. Positive fibres formed a dense network to surround hepatocytes. The present immunoelectron microscopic study has confirmed that neuropeptide Y-positive terminals are closely apposing to hepatocytes. Ontogenetically, neuropeptide Y-positive fibres were first found in embryonic liver of 19-day-old mice. Positive fibres increased with age and the highest peak was seen one week after birth. This ontogenetic pattern has suggested that neuropeptide Y plays a certain role in developing liver.

Effect of substituted benzyl chrysanthemates on sodium and potassium currents in the crayfish giant axon

Abstract The effects of substituted benzyl (1 R )- trans -chrysanthemates on sodium and potassium currents were studied in voltage-clamped crayfish giant axons. The o -SO 2 Me derivative decelerated the rate of the falling phase of the action potential (type A effect), and the p -SO 2 Me derivative elevated the depolarizing afterpotential (type B effect). The o -SO 2 Me derivative inhibited the inactivation of sodium channels. The time constant of the inactivation of sodium channels affected by the ortho derivative was 5–10 times that of the unaffected channels. The fraction of channels affected compared to the total sodium channels progressively increased during the administration of the drug. Voltage-sensitive potassium channels also were blocked progressively. The p -SO 2 Me derivative markedly slowed down the inactivation and induced a large residual current during depolarization. The time constant of the inactivation of sodium channels affected by this drug was 50–100 times that in control axons. Upon repolarization, a large tail current was observed, indicating that the restoration of gating groups responsible for the activation to the nonconducting position on repolarization also was suppressed in some affected channels. The m -CN derivative, which had a mixed effect of both types A and B on the action potential, caused both moderate slowing of the inactivation of some sodium channels and induction of the residual current. During the administration of this drug, a small tail current was observed upon step repolarization. We concluded that the type A effect on action potential is due to moderate slowing of the inactivation of the sodium channels and blocking of some fraction of voltage-sensitive potassium channels, and that the type B effect is due to extremely slow inactivation of sodium channels and the slowing of closing kinetics of the activation gate upon repolarization affected by drugs of this group.

Increases in K+ conductance and Ca2+ influx under high glucose with suppressed Na+/K+-pump activity in rat myelinated nerve fibers.

To test the combined effect of high glucose and decreased Na+/K+-pump activity, a condition which closely mimics the diabetic state, on nerve ionic currents, changes in action potential and membrane current induced by high glucose in the presence of ouabain were investigated using voltage clamp analysis in rat single myelinated nerve fibers. In the presence of 0.1 mM ouabain, 30 mM glucose caused a progressive increase in the delayed K+ current as well as persistent decreases in action potential and Na+ current, suggesting that Na+/K+ pump plays an important role in preventing the increase in the K+ current. The latter increase was suppressed by a blocker of Ca2+-activated K+ channels. Two types of voltage-dependent Ca2+ channel blockers (L and N-type) as well as a Na+/Ca2+-exchange blocker diminished the ouabain-induced increase in K+ conductance. These results suggest that high glucose with suppressed Na+/K+ pump activity might induce an increase of Ca2+ influx through either Ca2+channels or reverse Na+/Ca2+-exchange, possibly leading to the elevation of Ca2+-activated voltage-dependent K+ channels. Both a decrease in inward Na+ current and an increase in K+ conductance may result in decreased nerve conduction. In addition, a possible increase of axoplasmic Ca2+concentration may lead to axonal degeneration. These results provide a clue for understanding the pathophysiologic mechanism of diabetic neuropathy.

A possible role of the ATP-sensitive potassium ion channel in determining the duration of spike-bursts in mouse pancreatic β-cells

The pancreatic beta-cell displays an electrical activity consisting of spike bursts and silent phases at glucose concentrations of about 10 mM. The mechanism of initial depolarization induced by glucose is well defined. However, the mechanism inducing the silent phase has not been fully elucidated. In the present study, the possibility of involvement of ATP-sensitive K+ channels in repolarization was examined using the patch-clamp technique in the cell-attached recording configuration. Ouabain (0.1 mM), an inhibitor of Na+/K+-ATPase, caused a complete suppression of ATP-sensitive K+ channel activity followed by typical biphasic current deflections, which were due to action potentials. The channel activity was also inhibited by removal of K+ from a perifusion solution. Furthermore, the activity of ATP-sensitive K+ channels was markedly inhibited either by replacement of external NaCl with LiCl or by addition of amiloride (0.2 mM), a blocker of Na+/H+ antiport. Addition of L-type Ca2+ channel blockers such as Nifedipine for Mn2+ induced the complete suppression of K+ channel activity. These findings strongly suggest that a fall in ATP consumption results in sustained depolarization, and that the repolarizations interposed between spike-bursts under normal ionic conditions are due to the periodical fall of ATP concentration brought about by periodical acceleration of ATP consumption at Na+/K+-pumps. It is concluded that the elevation of intracellular Na+ concentration as a consequence of accelerated Na+/Ca2+-countertransport during the period of spike-burst enhances ATP consumption, leading to a fall in ATP concentration which is responsible for termination of spike-burst and initiation of repolarization.