Kari R. Hoyt

Nitric oxide modulates NMDA-induced increases in intracellular Ca2+ in cultured rat forebrain neurons

We studied the effects of nitric oxide (NO) and the NO-releasing agents sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and isosorbide dinitrate (ISDN) on N-methyl-D-aspartate (NMDA)-induced increases in intracellular Ca2+ ([Ca2+]i), whole-cell patch-clamp currents and on glutamate-stimulated [3H]dizocilpine binding. NO and agents that release NO partially inhibit increases in [Ca2+]i at concentrations between 1 microM and 1 mM. These agents also decrease [Ca2+]i changes produced by kainate and potassium, but to a smaller extent. As the effects of NO are still present following alkylation of the redox modulatory site on the NMDA receptor this action of NO is probably not a consequence of oxidation of the redox site. In contrast to SNP, ISDN does not inhibit NMDA-induced whole cell patch-clamp currents suggesting that NO modulates [Ca2+]i via perturbation of a Ca2+ homeostatic process. Furthermore, SNP may have a direct action on the NMDA receptor complex in addition to the generation of NO. 8-Bromo-cGMP does not mimic the inhibitory effect of NO suggesting that this effect is not the result of NO stimulation of neuronal cGMP production. As the production of NO in neurons is dependent on increases in [Ca2+]i associated with NMDA receptor activation, these data suggest that NO-mediated decreases in [Ca2+]i may represent a novel feedback inhibitory mechanism for NO production in the brain.

Mechanisms of dopamine-induced cell death in cultured rat forebrain neurons: interactions with and differences from glutamate-induced cell death.

Injury to the brain, whether by ischemia or trauma, results in the uncontrolled release of many neurotransmitters, including glutamate and dopamine. Both of these neurotransmitters are neurotoxic in high concentrations, and the oxidative stress caused by reactive oxygen species generation has been implicated in the mechanism of neurotoxicity. In this study, we used cultured rat forebrain neurons to characterize cell death caused by exposure to dopamine and/or glutamate and to investigate potential acute mechanisms of toxicity. Dopamine exposure (250 microM for 2 h) reduced cell viability to 34. 3 +/- 5.5% of untreated control 20 h later and increased the number of neurons with apoptotic morphology. The antioxidant N-acetylcysteine (100 microM) inhibited dopamine-induced toxicity and prevented the covalent binding of dopamine quinones to protein. In contrast, glutamate toxicity lacked the hallmark characteristics of apoptosis. When neurons were exposed successively to sublethal concentrations of dopamine and glutamate, cell viability at 20 h was reduced to 62.3 +/- 5.2% of untreated control. Apoptosis was not evident, and N-acetylcysteine blocked the potentiating effect of dopamine on glutamate-induced toxicity. We used single-cell fluorescence assays to measure changes in intraneuronal glutathione, intraneuronal Ca2+, mitochondrial membrane potential, and DNA integrity as potential acute inducers of neuronal injury. While changes in these parameters could be demonstrated, none were identified as the sole acute inducer of cell death caused by dopamine. In summary, we have characterized a number of neuronal responses to lethal dopamine injury. Also, we have demonstrated that dopamine and glutamate can interact in vitro to potentiate cell death and that the potentiation appears to be induced by oxidative stress.

Characterization of Hydrogen Peroxide Toxicity in Cultured Rat Forebrain Neurons

We investigated the ability of hydrogen peroxide (H2O2) to cause apoptotic cell death in cultured rat forebrain neurons and the potential mechanisms by which oxidative stress triggers delayed neuronal death. H2O2 (25 μM for 5 min) reduced cell viability to 34.5 ± 8.3% of untreated controls 20 h after exposure, and resulted in a significant proportion of neurons which exhibited apoptotic nuclear morphology. Using single cell fluorescence assays, we measured H2O2-induced changes in DNA strand breaks, 2′7′ dichlorofluorescin fluorescence, reduced glutathione, intracellular free Ca2+, and mitochondrial membrane potential. DNA strand breaks in response to H2O2 were not evident immediately following exposure, but were increased 12h and 20h after exposure. Millimolar concentrations of H2O2 caused increases in the fluorescence of the oxidant-sensitive fluorescent dye, 2′7′-dichlorofluorescin. H2O2 treatment decreased reduced glutathione following 30 minutes of exposure using the fluorescent indicator, 5-chloromethylfluorescein diacetate, and increased intra-neuronal free Ca2+ levels in a subpopulation of neurons. Mitochondrial membrane potential, measured by rhodamine 123 localization was unaffected by 25 μH2O2, while higher concentrations of H2O2 (10 or 30 mM) depolarized mitochondria. These studies demonstrate that H2O2 is a potent and effective neurotoxin that produces oxidative stress, as well as apoptotic neuronal death

Trifluoperazine and dibucaine-induced inhibition of glutamate-induced mitochondrial depolarization in rat cultured forebrain neurones

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 μM) and dibucaine (50–100 μM) 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.

Alkalinization Prolongs Recovery from Glutamate-Induced Increases in Intracellular Ca2+ Concentration by Enhancing Ca2+ Efflux Through the Mitochondrial Na+/Ca2+ Exchanger in Cultured Rat Forebrain Neurons

Abstract: Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 µM, for 15 s)‐induced increase in intracellular Ca2+ concentration ([Ca2+]i) in indo‐1‐loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES‐buffered saline solution (HBSS) was 126 ± 30 s, whereas recovery time was 216 ± 19 s when the pH was increased to 8.5. Removal of extracellular Ca2+ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca2+ recovery by affecting intraneuronal Ca2+ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca2+ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p‐(trifluoromethoxyphenyl)hydrazone (FCCP; 750 nM). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential‐sensitive fluorescent indicator, 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolocarbocyanine iodide (JC‐1). We found that the effect of pH 8.5 HBSS on Ca2+ recovery was completely inhibited by the mitochondrial Na+/Ca2+ exchange inhibitor CGP‐37157 (25 µM). This suggests that increased mitochondrial Ca2+ efflux via the mitochondrial Na2+/Ca2+ exchanger is responsible for the prolongation of [Ca2+]i recovery caused by alkaline pH following glutamate exposure.

The role of intracellular Na+ and mitochondria in buffering of kainate-induced intracellular free Ca2+ changes in rat forebrain neurones

1 We have examined the mechanisms by which cultured central neurones from embryonic rat brain buffer intracellular Ca2+ loads following kainate receptor activation using fluorescent indicators of [Ca2+]i and [Na+]i. 2 Stimulation of cultured forebrain neurones with 100 μm kainate produced a rapid increase in [Ca2+]i that displayed a variable rate of recovery. Kainate also increased [Na+]i with a response that was slightly slower in onset and markedly slower in recovery. 3 The recovery of [Ca2+]i to baseline was not very sensitive to the [Na+]i. The magnitude of the increase in [Na+]i in response to kainate did not correlate well with the [Ca2+]i recovery time, and experimental manipulations that altered [Na+]i did not have a large impact on the rate of recovery of [Ca2+]i. 4 The recovery of [Ca2+]i to baseline was accelerated by the mitochondrial Na+‐Ca2+ exchange inhibitor CGP‐37157, suggesting that the recovery rate is influenced by release of Ca2+ from a mitochondrial pool and also that variation in the recovery rate is related to the extent of mitochondrial Ca2+ loading. Kainate did not alter the mitochondrial membrane potential. 5 These studies reveal that mitochondria have a central role in buffering neuronal [Ca2+]i changes mediated by non‐N‐methyl‐D‐aspartate (NMDA) glutamate receptors, and that the variation in recovery times following kainate receptor activation reflects a variable degree of mitochondrial Ca2+ loading. However, unlike NMDA receptor‐mediated Ca2+ loads, kainate receptor activation has minimal effects on mitochondrial function.

Cyclothiazide modulates AMPA receptor-mediated increases in intracellular free Ca2+ and Mg2+ in cultured neurons from rat brain

Abstract: We investigated the modulation of (±)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐induced increases in intracellular free Ca2+ ([Ca2+]i) and intracellular free Mg2+ ([Mg2+]i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA‐induced changes in [Ca2+]i were increased by 0.3–100 µM cyclothiazide, with an EC50 value of 2.40 µM and a maximum potentiation of 428% of control values. [Ca2+]i responses to glutamate in the presence of N‐methyl‐d‐aspartate (NMDA) receptor antagonists were also potentiated by 10 µM cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non‐NMDA receptors. Almost all neurons responded with an increase in [Ca2+]i to both kainate and AMPA in the absence of extracellular Na+, and these Na+‐free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC50 value of 12.0 µM. Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA‐ and glutamate‐induced increases in [Mg2+]i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non‐NMDA glutamate receptors and may provide the basis for the increase in non‐NMDA receptor‐mediated excitotoxicity produced by cyclothiazide.

Localization of D1 dopamine receptors on live cultured striatal neurons by quantitative fluorescence microscopy

Single neurons in culture express a heterogeneity of neurotransmitter receptor subtypes. The study of the effects of neurotransmitters on neuronal function is complicated by this heterogeneity. It would therefore be useful to be able to identify live neurons that express the receptors of interest and then use these neurons for functional studies. We have used quantitative fluorescence microscopy to identify single live striatal neurons that express D1 dopamine receptors. The binding of the fluorescent D1 dopamine receptor antagonist bodipy-SCH 23390 was measured in 2-3-week-old primary striatal cultures derived from fetal rats (embryonic day 18). Binding of bodipy-SCH 23390 to live neurons was displaced by (+)-butaclamol, dopamine or SCH 23390, indicating that it specifically labelled D1 dopamine receptors. However, the fraction of bodipy-SCH 23390 binding that was specific varied substantially among individual neurons indicating heterogeneity of D1 dopamine receptor expression. Interestingly, bodipy-SCH 23390 also specifically labelled discrete spots of receptors on the neuronal processes. This technique should prove useful in the study of the effects of dopaminergic drugs on neuronal function in primary culture.

Intracellular Signalling in Glutamate Excitotoxicity

The excitatory neurotransmitter glutamate is a potent and effective neurotoxin. When applied in vitro, a. brief exposure to a moderate concentration of glutamate is sufficient to kill neurons.1,2 In vivo, glutamate-induced neuronal injury probably contributes to damage that results from cerebrovascular accidents and trauma.2–4 A number of important studies have characterized the temporal and pharmacological characteristics of glutamate excitotoxicity in vitro.5–6 It is now clear that glutamate-induced activation of N-methyl-D-aspartate (NMDA) receptors for about 5 minutes is sufficient to kill neurons, and that death is expressed within 24 hours of glutamate application. Activation of non-NMDA receptors by, for example, kainate requires exposures of more than 30 minutes; death ensues over a similar time frame.