Sadahiro Iwabuchi

Selective blockade of astrocytic glutamate transporter GLT-1 with dihydrokainate prevents neuronal death during ouabain treatment of astrocyte/neuron cocultures

Glutamate (Glu) is a major excitatory neurotransmitter of the mammalian central nervous system and under normal conditions plays an important role in information processing in the brain. Therefore, extracellular Glu is subject to strong homeostasis. Astrocytes in the brain have been considered to be mainly responsible for the clearance of extracellular Glu. In this study, using mixed neuron/astrocyte cultures, we investigated whether astrocytic Glu transporter GLT‐1 is crucial to the survival of neurons under various conditions. Treatment of the mixed cultures with a low concentration of Glu did not produce significant death of neurons. However, cotreatment with dihydrokainate (DHK), a specific blocker of GLT‐1, resulted in significant neuronal death that was suppressed by an antagonist of N‐methyl‐D‐aspartate (NMDA) receptors. These results suggested that astrocytic GLT‐1 participated in the clearance of extracellular Glu and protected neurons from NMDA receptor‐mediated toxicity. When the cultures were treated with ouabain, an inhibitor of Na+/K+‐ATPase, a low concentration of Glu resulted in massive neuronal death that was also suppressed by cotreatment with an antagonist of NMDA receptors. In this case, however, cotreatment with DHK significantly protected neurons from death, suggesting that GLT‐1 was responsible for the death of neurons. The present study provides evidence suggesting that astrocytes use their Glu transporter GLT‐1 to protect neurons from Glu toxicity, but, ironically, also use GLT‐1 to kill neurons through Glu toxicity depending on their status. GLIA 40:337–349, 2002.

Functional significance of the negative-feedback regulation of ATP release via pannexin-1 hemichannels under ischemic stress in astrocytes.

The opening of pannexin-1 (Px1) hemichannels is regulated by the activity of P2X(7) receptors (P2X(7)Rs). At present, however, little is known about how extracellular ATP-sensitive P2X(7)Rs regulates the opening and closure of Px1 hemichannels. Several lines of evidence suggest that P2X(7)Rs are activated under pathological conditions such as ischemia, resulting in the opening of Px1 hemichannels responsible for the massive influx of Ca(2+) from the extracellular space and the release of ATP from the cytoplasm, leading to cell death. Here we show in cultured astrocytes that the suppression of the activity of P2X(7)Rs during simulated ischemia (oxygen/glucose deprivation, OGD) resulted in the opening of Px1 hemichannels, leading to the enhanced release of ATP. In addition, the suppression of the activity of P2X(7)Rs during OGD resulted in a significant increase in astrocytic damage. Both the P2X(7)Rs suppression-induced enhancement of the release of ATP and cell damage were reversed by co-treatment with blockers of Px1 hemichannels, suggesting that suppression of the activity of PX(7)Rs resulted in the opening of Px1 hemichannels. All these findings suggested the existence of a negative-feedback loop regulating the release of ATP via Px1 hemichannels; ATP-induced suppression of ATP release. The present study indicates that ATP, released through Px1 hemichannels, activates P2X(7)Rs, resulting in the closure of Px1 hemichannels during ischemia. This negative-feedback mechanism, suppressing the loss of cellular ATP and Ca(2+) influx, might contribute to the survival of astrocytes under ischemic stress.

Relationship between the activation of cyclic AMP responsive element binding protein and ischemic tolerance in the penumbra region of rat cerebral cortex.

Application of a brief period of ischemia, i.e. preconditioning treatment of the middle cerebral artery territory, has been known to produce ischemic tolerance, reducing cerebral infarction volume in the penumbra region after lethal ischemia. However, little is known about the molecular mechanisms responsible for preconditioning-induced ischemic tolerance. In the present study, we examined the difference in the phosphorylation pattern of cyclic AMP responsive element binding protein (CREB) after 1 h of focal cerebral ischemia between preconditioned and non-preconditioned rats by immunohistochemistry and Western blotting. The phosphorylation of CREB in the penumbra region was more rapidly enhanced in the preconditioned rats than in the non-preconditioned rats after 1 h of ischemia. The result suggested that the immediate enhancement in the phosphorylation of CREB in the penumbra region prevented the spread of infarction in the preconditioned rats.

Inducible astrocytic glucose transporter-3 contributes to the enhanced storage of intracellular glycogen during reperfusion after ischemia.

Glucose is a necessary source of energy to sustain cell activities and homeostasis in the brain, and enhanced glucose transporter (GLUT) activities are protective of cells during energy depletion including brain ischemia. Here we investigated whether and if so how the astrocytic expression of GLUTs crucial for the uptake of glucose changes in ischemic conditions. Under physiological conditions, cultured astrocytes primarily expressed GLUT1, and GLUT3 was only detected at extremely low levels. However, exposure to ischemic stress increased the expression of not only GLUT1 but also GLUT3. During ischemia, cultured astrocytes significantly increased production of the transcription factor nuclear factor-κB (NF-κB), leading to an increase in GLUT3 expression. Moreover, astrocytic GLUT3 was responsible for the enhanced storage of intracellular glucose during reperfusion, resulting in increased resistance to lethal ischemic stress. These results suggested that astrocytes promptly increase GLUT3 production in situations such as ischemia, and much glucose is quickly taken up, possibly contributing to the protection of astrocytes from ischemic damage.

Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia.

Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. Ischemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2h after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes.

Photolytic flash-induced intercellular calcium waves using caged calcium ionophore in cultured astrocytes from newborn rats

Waves of elevated intracellular free calcium that propagate between neighboring astrocytes are important for the intercellular communication between astrocytes as well as between neurons and astrocytes. However, the mechanisms responsible for the initiation and propagation of astrocytic calcium waves remain unclear. In this study, intercellular calcium waves were evoked by focal photolysis of a caged calcium ionophore (DMNPE-caged Br A23187) in cultured astrocytes from newborn rats. The focal photolysis of the caged compound resulted in the increase in intracellular calcium in a single astrocyte, and this increase then propagated to neighboring astrocytes. We also analyzed the spatiotemporal characteristics of the intercellular calcium waves, and estimated the propagation pathways for them. The method using a caged calcium ionophore described in this study provides a new in vitro model for the analysis of intercellular calcium waves.

Possible Involvement of Extracellular ATP-P2Y Purinoceptor Signaling in Ischemia-induced Tolerance of Astrocytes in Culture

Extracellular adenosine 5′-triphosphate (ATP) activates specific G protein-coupled purinoceptors (P2Y), and ATP-P2Y signaling pathways induces intracellular Ca2+ mobilization resulting in changes in the gene expression of a variety of proteins in astrocytes. This study investigated whether the exposure of cultured astrocytes to sublethal ischemia produced resistance to subsequent lethal ischemic stress, and if so, whether the extracellular ATP-P2Y signaling pathways were responsible for the tolerance. Ischemia-like insults, sublethal oxygen-glucose deprivation (sOGD), produced tolerance to subsequent lethal OGD stress in cultured astrocytes. Early during reperfusion after sOGD, the amount of extracellular ATP and the expression of both P2Y1 and P2Y2 receptors were increased, leading to enhanced activation of the extracellular ATP-P2Y signaling pathways. The occurrence of intracellular spontaneous Ca2+ oscillations was also increased. In addition, sOGD treatment enhanced the expression of the phosphorylated form of extracellular signal-regulated protein kinases 1 and 2 (p-ERK 1/2), and treatment with an inhibitor of ERK significantly attenuated the sOGD-induced ischemic tolerance of astrocytes.

Rhythmic Fluctuations in the Concentration of Intracellular Mg2+ in Association with Spontaneous Rhythmic Contraction in Cultured Cardiac Myocytes

Magnesium ions (Mg2+) play a fundamental role in cellular function, but the cellular dynamic changes of intracellular Mg2+ remain poorly delineated. The present study aims to clarify whether the concentration of intracellular Mg2+ possibly changes cyclically in association with rhythmic contraction and intracellular Ca2+ oscillation in cultured cardiac myocytes from neonatal rats. To do this, we performed a noise analysis of fluctuations in the concentration of intracellular Mg2+ in cardiac myocytes. The concentration was estimated by loading cells with either Mg‐fluo4/AM or KMG‐20/AM. Results revealed that the intensity of Mg‐fluo‐4 or KMG‐20 fluorescence fluctuated cyclically in association with the rhythmic contraction of cardiac myocytes. In addition, the simultaneous measurement of Fura2 and Mg‐fluo‐4 fluorescence revealed phase differences between the dynamics of the two signals, suggesting that the cyclic changes in the Mg‐fluo‐4 or KMG‐20 fluorescent intensity actually reflected the changes in intracellular Mg2+. The complete termination of spontaneous rhythmic contractions did not abolish Mg2+ oscillations, suggesting that the rhythmic fluctuations in intracellular Mg2+ did not result from mechanical movements. We suggest that the concentration of intracellular Mg2+ changes cyclically in association with spontaneous, cyclic changes in the concentration of intracellular Ca2+ of cardiac myocytes. A noise analysis of the fluctuation of subtle changes in fluorescence intensity could contribute to the elucidation of novel functional roles of Mg2+ in cells.

Oxygen-glucose deprivation-induced enhancement of extracellular ATP-P2Y purinoceptors signaling for the propagation of astrocytic calcium waves

Waves of elevated intracellular free Ca(2+) that propagate between neighboring astrocytes (Ca(2+) waves) are important for the communication among astrocytes. We have previously revealed that focal photolysis of a caged calcium ionophore results in an increase in the concentration of intracellular Ca(2+) in the target astrocytes, then the increase propagates to neighboring astrocytes through gap junctions. The extracellular ATP-purinoceptors signaling pathways are not primarily responsible for the propagation of the photolytic flash-induced Ca(2+) waves. Here we examined whether and if so how the dynamics of Ca(2+) waves changed after treatment with sublethal simulated ischemia; oxygen-glucose deprivation (OGD). OGD treatment increased the astrocytic expression of P2Y(1) and P2Y(2) receptors early during reperfusion, resulting in an increase in the propagating waves speed. In contrast, the expression of a gap junction protein was not changed significantly by the OGD suggesting that the extracellular ATP-P2Y receptors signaling pathways were preferentially enhanced after OGD. The present method to induce Ca(2+) waves by focal photolysis of a caged calcium ionophore may provide a valuable tool with which to analyze glial Ca(2+) waves under not only normal but also pathologic conditions.

Negative-Feedback Regulation of ATP Release During Ischemia in Cardiac Myocytes

Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes.