dizocilpine-maleate and 5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine

Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase.

Topics: 2-Amino-5-phosphonovalerate; Aniline Compounds; Animals; Astrocytes; Benzimidazoles; Calcium; Calcium Channel Blockers; Carbocyanines; Cells, Cultured; Dizocilpine Maleate; Endoplasmic Reticulum; Inositol 1,4,5-Trisphosphate Receptors; Ions; Kynurenic Acid; Membrane Potential, Mitochondrial; N-Methylaspartate; Protein Subunits; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; RNA, Small Interfering; Xanthenes

This study is designed to investigate the effect of morphine on glutamate-induced toxicity of primary rat neonatal astrocytes. Glutamate decreases the intracellular GSH level, and thereby induces cytolysis of astrocytes and C6 glial cells accompanied by apoptotic features. Glutamate-induced cytotoxicity is protected by morphine and antioxidants such as GSH and NAC, whereas MK-801, an antagonist of glutamate receptor NMDA does not protect astrocytes against glutamate toxicity. Also, morphine antagonist, naloxone, as well as selective ligands for opioid receptor subtypes, including DAMGO, DPDPE, and U69593, do not inhibit the protective effect of morphine on glutamate-induced cytotoxicity. Morphine significantly prevents the depletion of GSH by glutamate and thereby inhibits the generation of H2O2 in a dose-dependent manner. Furthermore, morphine prevents the change of mitochondrial permeability transition by glutamate. Taken together, we suggest that morphine protects the primary rat neonatal astrocytes from glutamate toxicity via modulation of intracellular redox status.

Topics: Acetylcysteine; Animals; Animals, Newborn; Apoptosis; Astrocytes; Benzeneacetamides; Benzimidazoles; Bisbenzimidazole; Carbocyanines; Cell Line, Tumor; Cell Nucleus; Cell Survival; Cells, Cultured; Dizocilpine Maleate; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Glutamic Acid; Glutathione; Hydrogen Peroxide; Ion Channels; Microscopy, Fluorescence; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Morphine; Naloxone; Narcotic Antagonists; Neuroglia; Oxidation-Reduction; Oxidative Stress; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Rhodamine 123

Progesterone modulates gamma-aminobutyric acid and excitatory amino acid neurotransmitter systems and has neuroprotective properties in models of hypoxia-ischemia. This study examined the in vitro effects of allopregnanolone, the active progesterone metabolite, in models of N-methyl-D-aspartate (NMDA)-induced necrosis and apoptosis. Cultured NT2 neurons were exposed to 1 mM NMDA. Lactate dehydrogenase (LDH) release was measured 24 h later. NMDA at a concentration of 1 mM produced a 39 +/- 19% release of total LDH. Exposure to 10 microM allopregnanolone prior to NMDA exposure reduced LDH release by 51% (P = 0.0028). NMDA stimulated apoptotic cell changes defined by terminal dUTP nick-end labeling (TUNEL) and 5,5', 6,6'-tetrachloro-1,1,3,3'-tetra ethlybenzimidazolycarbocyanide iodide staining were reduced to baseline values by both 10 microM allopregnanolone and 100 microM MK-801. Pretreatment with allopregnanolone (0-10 microM) reduced the percentage of TUNEL-positive cells in a dose-dependent manner (EC(50) = 2.7 +/- 0.1 nM). Physiologic concentrations of allopregnanolone provided protection against both necrotic and apoptotic injury induced by NMDA excitotoxicity.

Topics: Apoptosis; Asphyxia Neonatorum; Benzimidazoles; Carbocyanines; Cell Count; Cell Survival; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Fluorescent Dyes; Humans; Hypoxia-Ischemia, Brain; In Situ Nick-End Labeling; Infant, Newborn; L-Lactate Dehydrogenase; Membrane Potentials; Mitochondria; N-Methylaspartate; Neurons; Neuroprotective Agents; Neurotoxins; Pregnancy; Pregnanolone; Progesterone; Tumor Cells, Cultured

1. Glutamate receptor activation has been previously shown to result in mitochondrial depolarization and activation of the mitochondrial permeability transition pore in cultured neurones. In this study, we characterized the effects of two putative permeability transition inhibitors, namely trifluoperazine and dibucaine, on mitochondrial depolarization in rat intact, cultured forebrain neurones. 2. Permeability transition was monitored by following mitochondrial depolarization in neurones loaded with the mitochondrial membrane potential-sensitive fluorescent indicator, JC-1. Trifluoperazine (10 20 microM) and dibucaine (50-100 microM) inhibited or delayed the onset of glutamate-induced permeability transition. 3. We also investigated the effects of trifluoperazine and dibucaine on neuronal recovery from glutamate-induced Ca2+ loads. Trifluoperazine affected Ca2+ recovery in a manner similar to the mitochondrial Na+/Ca2+ exchange inhibitor, CGP-37157, while dibucaine had no apparent effect on Ca2+ recovery. Therefore, inhibition of permeability transition does not appear to be involved in Ca2+ recovery from glutamate-induced Ca2+ loads. 4. Trifluoperazine and dibucaine did not inhibit [3H]-dizocilpine binding at the concentrations that prevented mitochondrial depolarization. 5. These studies suggest that trifluoperazine and dibucaine inhibit permeability transition in intact neurones. Trifluoperazine also appears to inhibit mitochondrial Na+/Ca2+ exchange. These drugs should prove to be valuable tools in the further study of the role of mitochondrial permeability transition in glutamate-induced neuronal death.

Topics: Animals; Benzimidazoles; Calcium; Carbocyanines; Cells, Cultured; Clonazepam; Dibucaine; Dizocilpine Maleate; Fluorescent Dyes; Fluorometry; Glutamic Acid; Membrane Potentials; Mitochondria; Neurons; Prosencephalon; Rats; Rats, Sprague-Dawley; Sodium-Calcium Exchanger; Thiazepines; Trifluoperazine