Koichi Kawahara

Nitric Oxide Produced During Sublethal Ischemia Is Crucial for the Preconditioning-Induced Down-Regulation of Glutamate Transporter GLT-1 in Neuron/Astrocyte Co-Cultures

In the brain, prior sublethal ischemia (preconditioning, PC) produces tolerance of neurons to subsequent lethal ischemia. This study aims at elucidating whether and how nitric oxide (NO) produced during PC is involved in the PC-induced ischemic tolerance of neurons in neuron/astrocyte co-cultures. The rise in the extracellular concentration of glutamate during ischemia caused by the reversed uptake of glutamate (Glu) by the astrocytic Glu transporter GLT-1 was markedly suppressed by the prior PC treatment, but the suppression was reversed by treatment with an inhibitor of nitric oxide synthase (NOS) during PC. Immunocytochemical and Western blot analyses demonstrated that the expression of GLT-1 was down-regulated after the PC insult, and this down-regulation was also antagonized by treatment with NOS inhibitors during PC. Here we show that nNOS-derived NO produced during PC was crucial for the down-regulation of astrocytic GLT-1, and this down-regulation coincided with an increased survival rate of neurons.

Reversed Actrocytic GLT-1 during Ischemia is Crucial to Excitotoxic Death of Neurons, but Contributes to the Survival of Astrocytes themselves

During ischemia, the operation of astrocytic/neuronal glutamate transporters is reversed and glutamate and Na+ are co-transported to the extracellular space. This study aims to investigate whether this reversed operation of glutamate transporters has any functional meanings for astrocytes themselves. Oxygen/glucose deprivation (OGD) of neuron/astrocyte co-cultures resulted in the massive death of neurons, and the cell death was significantly reduced by treatment with either AP5 or DHK. In cultured astrocytes with little GLT-1 expression, OGD produced Na+ overload, resulting in the reversal of astrocytic Na+/Ca2+-exchanger (NCX). The reversed NCX then caused Ca2+ overload leading to the damage of astrocytes. In contrast, the OGD-induced Na+ overload and astrocytic damage were significantly attenuated in PACAP-treated astrocytes with increased GLT-1 expression, and the attenuation was antagonized by treatment with DHK. These results suggested that the OGD-induced reversal of GLT-1 contributed to the survival of astrocytes themselves by releasing Na+ with glutamate via reversed GLT-1.

Ruthenium red-induced transition from ventricular fibrillation to tachycardia in isolated rat hearts:: possible involvement of changes in mitochondrial calcium uptake

INTRODUCTIONnVentricular tachycardia (VT) is considered to be the most common precursor of ventricular fibrillation (VF) and sudden cardiac death. However, the mechanisms underlying the transition from VT to VF remain unclear despite more than a century of study. Here, we investigated whether perfusion of the heart with blockers of mitochondrial Ca(2+) uniporter changed the macrodynamics of the heart between VT and VF.nnnMETHODSnThe experiments were performed using Langendorff perfused isolated rat hearts in which left ventricular pressure (LVP) and left ventricular cardiomyogram (LVCMG) were measured. Sustained VT or VF was induced by burst pacing of the left ventricular muscles.nnnRESULTSnDuring pacing-induced sustained VF, perfusion of the heart with ruthenium red (RR) or Ru 360, blockers of mitochondrial Ca(2+) uniporter, resulted in the reversible conversion of VF to VT. In contrast, during pacing-induced sustained VT, perfusion of the heart with spermine, an activator of mitochondrial Ca(2+) uptake, resulted in the reversible conversion of VT to VF, and the effect was antagonized by cotreatment with RR. In addition, RR-induced conversion of VF to VT was antagonized by cotreatment with S(-)-Bay K8644 (Bay K), an activator of L-type Ca(2+) channels, suggesting that the inactivation of L-type Ca(2+) channels was responsible for the RR-induced effect on the macrodynamics of hearts. In fact, perfusion with verapamil, an antagonist of L-type Ca(2+) channels, during pacing-induced sustained VF, resulted in the conversion of VF to VT.nnnCONCLUSIONnThis study demonstrated that perfusion of isolated rat hearts with blockers of Ca(2+) uptake by mitochondria resulted in the reversible conversion of pacing-induced sustained VF to VT, suggesting that changes in mitochondrial Ca(2+) uptake were possibly involved in the transition between VT and VF.

Nitric Oxide Produced During Ischemia Is Toxic but Crucial to Preconditioning-Induced Ischemic Tolerance of Neurons in Culture

The present study investigated the roles of nitric oxide (NO) in preconditioning (PC)-induced neuronal ischemic tolerance in cortical cultures. Ischemia in vitro was simulated by subjecting cultures to both oxygen and glucose deprivation (OGD). A sublethal OGD (PC) significantly increased the survival rate of neurons when cultures were exposed to a lethal OGD 24 h later. Both the inhibition of nitric oxide synthase (NOS) and scavenging of NO during PC significantly attenuated the PC-induced neuronal tolerance. In addition, exposure to an NO donor emulated the PC. In contrast, the inhibition of NOS and the scavenging of NO during lethal OGD tended to increase the survival rate of neurons. This study suggested that NO produced during ischemia was fundamentally toxic, but critical to the development of PC-induced neuronal tolerance.

Fluctuations of Contraction Rhythm During Simulated Ischemia/Reperfusion in Cultured Cardiac Myocytes from Neonatal Rats

Cardiac ischemia results in a rapid decrease of intracellular pH and in the rise of intracellular Ca 2+, changes that have been shown to reduce intercellular communication via gap junctions (GJ) between cardiac myocytes. Ischemia also results in electrical instability probably caused by the reduced GJ permeability contributing to an increased vulnerability to arrhythmias. This study aims at elucidating whether the fluctuations of contraction rhythm of spontaneously beating cardiac myocytes in culture changes during simulated ischemia/reperfusion. The coefficient of variation (CV) of contraction intervals, reflecting the fluctuation of contraction rhythm, increased significantly during simulated ischemia/reperfusion. However, the contraction rhythm of the cardiac myocytes in an aggregate remained synchronized during simulated ischemia/reperfusion. In contrast, pharmacological blockade of GJ with 12-doxyl stearic acid, a blocker of GJ permeability, resulted in the de-synchronization of contraction rhythm and in an increase in the CV of contraction intervals in normoxic conditions. The present findings lead to the suggestion that GJ remained open during simulated ischemia/reperfusion, and that a mechanism other than electrical uncoupling between myocytes contributed to the observed increase in the fluctuation of beating rhythm during ischemia.

Functional significance of the preconditioning-induced down-regulation of glutamate transporter GLT-1 in neuron/astrocyte co-cultures.

In the brain, prior sublethal ischemia (preconditioning, PC) is known to produce tolerance of neurons to subsequent lethal ischemia. This study aims at elucidating what alterations were induced in neurons and/or astrocytes by PC treatment. The rise in the extracellular concentration of glutamate during ischemia was markedly suppressed by the prior PC treatment. Immunocytochemical and Western blot analyses demonstrated that the expression of the astrocytic glutamate transporter GLT-1 was transiently down-regulated after the PC insult. The PC insult possibly suppressed the neuron-derived factors up-regulating GLT-1. Here we show that PC-induced down-regulation of GLT-1 is crucial for the increased neuronal resistance to subsequent severe ischemic insult.

Microneurography in anesthetized rats for the measurement of sympathetic nerve activity in the sciatic nerve

Microneurography is widely used for the measurement of human peripheral sympathetic nerve activity (SNA) in conscious subjects by virtue of its low invasive nature, but has rarely been employed in animal experiments. Because the low invasive nature sometimes is very useful even in animal experiments, we tested its feasibility for the measurement of SNA in the sciatic nerve of the anesthetized rat, aiming in particular to establish a methodology for measurement. A tungsten microelectrode was inserted into the nerve exposed at the thigh level to detect the spontaneous, intermittent burst signal that is one of the main characteristics of compound SNA. Such signals were found in more than 70% of experiments after surgical operators became accustomed to the method. Whenever such signals were detected, electrical stimulation of the sympathetic chain resulted in induced action potentials detected with the microelectrode after a reasonable conduction period. The spikes were successfully reduced after administration of hexamethonium bromide, a sympathetic ganglion blocker. On the other hand, induced spikes were never observed when we failed to find the spontaneous, intermittent burst signal. The results demonstrate the feasibility of microneurography in evaluating SNA in anesthetized small animals, and that the spontaneous, intermittent burst signal may be sufficient to identify SNA.

Increased vulnerability to ischemia/reperfusion-induced ventricular tachyarrhythmias by pre-ischemic inhibition of nitric oxide synthase in isolated rat hearts

INTRODUCTIONnThe relationship between vulnerability to reperfusion-induced ventricular tachyarrhythmias, such as ventricular tachycardia (VT) and fibrillation (VF), and the endogenous activity of nitric oxide synthase (NOS) has not been well documented. The objective of the present study was to clarify whether the vulnerability to reperfusion-induced VT/VF changes with preishemic, sustained inhibition of NOS.nnnMETHODSnThe experiments were performed using Langendorff-perfused isolated rat hearts, in which left ventricular pressure (LVP) and left ventricular cardiomyograms (LVCMGs) were measured.nnnRESULTSnA pre-ischemic, sustained inhibition of NOS resulted in an increased vulnerability to reperfusion-induced VT/VF, and the increase was markedly attenuated by co-treatment with L-arginine or by post-ischemic treatment with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of tetrahydrobiopterin (BH(4)) synthesis. We then tried to elucidate whether nitric oxide (NO) and superoxide were produced during reperfusion, and ATP-sensitive potassium channels (K(ATP)), especially mitochondrial ATP-sensitive potassium channels (mitoK(ATP)), are involved in the increased vulnerability. Post-ischemic inhibition of NOS and treatment with a NO scavenger attenuated the increased vulnerability to reperfusion-induced VT/VF, but post-ischemic treatment with a superoxide scavenger did not. In addition, post-ischemic treatment with S-nitroso-N-acetyl-DL-penicillamine (SNAP), a NO donor, or with diazoxide, a selective opener of mitoK(ATP), increased the VT/VF duration during reperfusion. The increased vulnerability to VT/VF was attenuated by the treatment with a selective mitoK(ATP) blocker.nnnCONCLUSIONnThe results suggest that a pre-ischemic, sustained inhibition of NOS increases the vulnerability to reperfusion-induced VT/VF, and the NO-mitoK(ATP) pathway is one of the possible factors contributing to the increased vulnerability to VT/VF.

Increased resistance to nitric oxide cytotoxicity associated with differentiation of neuroblastoma-glioma hybrid (NG108-15) cells.

Nitric oxide (NO), synthesized by the enzyme nitric oxide synthase (NOS), acts as an intercellular messenger associated with various physiological and pathological events. In this study, we investigated whether there exits a difference in the vulnerability to NO-induced cytotoxicity between undifferentiated and differentiated NG108-15 cells, and if so, the mechanisms responsible for the difference. Following a 7- to 8-day exposure to dibutyryl cAMP (dbcAMP), NG108-15 cells exhibited a neuron-like morphology associated with the expression of the neuronal protein, synaptophysin, and with increased NADPH-d activity. Neuron-like differentiated NG108-15 cells acquired resistance to exogenously applied NO. This increased resistance to NO toxicity in differentiated cells was almost completely cancelled out by inhibiting the activity of superoxide dismutase (SOD), but not by inhibiting the activity of NOS. The present study suggested that the activity of SOD increased in parallel with the activity of NOS associated with differentiation and was crucial for the acquired resistance to NO toxicity in differentiated cells.

Rhythmic contraction and intracellular Ca2 + oscillatory rhythm in spontaneously beating cultured cardiac myocytes

Abstract Cultured cardiac myocytes from neonatal rats show spontaneous and rhythmic contractions. The intracellular concentration of free Ca2u2009+u2009 also changes rhythmically, associated with the rhythmic contraction of myocytes (Ca2u2009+u2009 oscillation). This study aims to elucidate whether spontaneous rhythmic contraction affects the dynamics of intracellular Ca2u2009+u2009 oscillation in cultured cardiac myocytes. In cultures at four days in vitro (4 DIV), spontaneous Ca2u2009+u2009 oscillation was synchronized among myocytes. Treatment of cultures with an uncoupler of Eu2009–u2009C coupling resulted in a cessation of the spontaneous contraction of cardiac myocytes, but did not abolish the Ca2u2009+u2009 oscillation. The intercellular synchronization of intracellular Ca2u2009+u2009 oscillation persisted, and both the intervals and the fluctuation of the oscillation tended to increase after the termination of rhythmic contraction. The present study demonstrated that mechanical factors associated with rhythmic contraction did not affect the intercellular synchronization of intracellular Ca2u2009+u2009 oscillation, but possibly contributed to the stability of the oscillatory rhythm.