Karen A. Willoughby

Calcium Influx Factor, Further Evidence It Is 5,6-Epoxyeicosatrienoic Acid

We present evidence in astrocytes that 5,6-epoxyeicosatrienoic acid, a cytochrome P450 epoxygenase metabolite of arachidonic acid, may be a component of calcium influx factor, the elusive link between release of Ca2+ from intracellular stores and capacitative Ca2+ influx. Capacitative influx of extracellular Ca2+ was inhibited by blockade of the two critical steps in epoxyeicosatrienoic acid synthesis: release of arachidonic acid from phospholipid stores by cytosolic phospholipase A2 and cytochrome P450 metabolism of arachidonic acid. AAOCF3, which inhibits cytosolic phospholipase A2, blocked thapsigargin-stimulated release of arachidonic acid as well as thapsigargin-stimulated elevation of intracellular free calcium. Inhibition of P450 arachidonic acid metabolism with SKF525A, econazole, orN-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide, a substrate inhibitor of P450 arachidonic acid metabolism, also blocked thapsigargin-stimulated Ca2+ influx. Nano- to picomolar 5,6-epoxyeicosatrienoic acid induced [Ca2+] i elevation consistent with capacitative Ca2+ influx. We have previously shown that 5,6-epoxyeicosatrienoic acid is synthesized and released by astrocytes. When 5,6-epoxyeicosatrienoic acid was applied to the rat brain surface, it induced vasodilation, suggesting that calcium influx factor may also serve a paracrine function. In summary, our results suggest that 5,6-epoxyeicosatrienoic acid may be a component of calcium influx factor and may participate in regulation of cerebral vascular tone.

Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons.

Abstract: Energy deficit after traumatic brain injury (TBI) may alter ionic homeostasis, neurotransmission, biosynthesis, and cellular transport. Using an in vitro model for TBI, we tested the hypothesis that stretch‐induced injury alters mitochondrial membrane potential (Δ?m) and ATP in astrocytes and neurons. Astrocytes, pure neuronal cultures, and mixed neuronal plus glial cultures grown on Silastic membranes were subjected to mild, moderate, and severe stretch. After injury, Δ?m was measured using rhodamine‐123, and ATP was quantified with a luciferin‐luciferase assay. In astrocytes, Δ?m dropped significantly, and ATP content declined 43‐52% 15 min after mild or moderate stretch but recovered by 24 h. In pure neurons, Δ?m declined at 15 min only in the severely stretched group. At 48 h postinjury, Δ?m remained decreased in severely stretched neurons and dropped in moderately stretched neurons. Intracellular ATP content did not change in any group of injured pure neurons. We also found that astrocytes and neurons release ATP extracellularly following injury. In contrast to pure neurons, Δ?m in neurons of mixed neuronal plus glial cultures declined 15 min after mild, moderate, or severe stretch and recovered by 24‐48 h. ATP content in mixed cultures declined 22‐28% after mild to severe stretch with recovery by 24 h. Our findings demonstrate that injury causes mitochondrial dysfunction in astrocytes and suggest that astrocyte injury alters mitochondrial function in local neurons.

Intracellular free calcium dynamics in stretch-injured astrocytes.

Abstract: We have previously developed an in vitro model for traumatic brain injury that simulates a major component of in vivo trauma, that being tissue strain or stretch. We have validated our model by demonstrating that it produces many of the posttraumatic responses observed in vivo. Sustained elevation of the intracellular free calcium concentration ([Ca2+]i) has been hypothesized to be a primary biochemical mechanism inducing cell dysfunction after trauma. In the present report, we have examined this hypothesis in astrocytes using our in vitro injury model and fura‐2 microphotometry. Our results indicate that astrocyte [Ca2+]i is rapidly elevated after stretch injury, the magnitude of which is proportional to the degree of injury. However, the injury‐induced [Ca2+]i elevation is not sustained and returns to near‐basal levels by 15 min postinjury and to basal levels between 3 and 24 h after injury. Although basal [Ca2+]i returns to normal after injury, we have identified persistent injury‐induced alterations in calcium‐mediated signal transduction pathways. We report here, for the first time, that traumatic stretch injury causes release of calcium from inositol trisphosphate‐sensitive intracellular calcium stores and may uncouple the stores from participation in metabotropic glutamate receptor‐mediated signal transduction events. We found that for a prolonged period after trauma astrocytes no longer respond to thapsigargin, glutamate, or the inositol trisphosphate‐linked metabotropic glutamate receptor agonist trans‐(1S,3R)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid with an elevation in [Ca2+]i. We hypothesize that changes in calcium‐mediated signaling pathways, rather than an absolute elevation in [Ca2+]i, is responsible for some of the pathological consequences of traumatic brain injury.

Ionizing radiation modulates vascular endothelial growth factor (VEGF) expression through multiple mitogen activated protein kinase dependent pathways.

We investigated the role of radiation-induced mitogen activated protein kinase (MAPK) pathway activity in the regulation of proliferation, cell survival and vascular endothelial growth factor (VEGF) production in primary astrocytes and in T9 and RT2 glioblastoma cells derived from Fisher 344 rats. In these cells, ionizing radiation (2 Gy) caused activation of the MAPK pathway which was blocked by specific inhibitor drugs. Blunting of radiation-induced MAPK activity weakly enhanced radiation-induced apoptosis 24 h after exposure in RT2 cells. Furthermore, blunting of MAPK activation weakly enhanced the ability of radiation to reduce RT2 cell growth in clonogenic growth assays. These findings argue that inhibition of MAPK signaling reduces proliferation and enhances cell killing by ionizing radiation in transformed astrocytes. Proliferation and survival of cancer cells has been linked in vivo to enhanced expression of angiogenic growth factors. Recently we demonstrated that the gene product of a novel rodent radiation-responsive gene, progression elevated gene 3 (PEG-3), could enhance vascular endothelial growth factor (VEGF) promoter activity in rodent fibroblasts, leading to increased VEGF protein levels and tumorigenic behavior in vivo. Thus PEG-3 and VEGF expression could be expected to directly correlate with the oncogenic potential of transformed cells. RT2 cells expressed more PEG-3 and VEGF protein than T9 cells, and were more tumorigenic in vivo than T9 cells. Radiation activated the PEG-3 promoter via MAPK signaling and ectopic over-expression of PEG-3 enhanced both basal MAPK activity and basal VEGF promoter activity. Basal MAPK activity partially correlated with basal VEGF promoter activity and VEGF protein levels in primary astrocytes, T9 and RT2 cells. Radiation increased the activity of the VEGF promoter and VEGF protein levels in primary astrocytes, T9 and RT2 cells which were dependent upon MAPK function. Furthermore, inhibition of AP-1 transcription factor signaling by dominant negative c-Jun (TAM67) also significantly reduced basal, and to a lesser extent radiation-induced, VEGF promoter function in RT2 cells. Collectively, our data demonstrate that radiation-induced MAPK signaling can both protect cells from radiation-induced cell death as well as enhance protein levels of pro-angiogenic factors such as VEGF. Enhanced VEGF expression in RT2 cells may be mediated via MAPK and JNK pathway signaling which converges upon the AP-1 transcription factor complex.

Stretch-Induced Injury of Cultured Neuronal, Glial, and Endothelial Cells Effect of Polyethylene Glycol–Conjugated Superoxide Dismutase

BACKGROUND AND PURPOSE There is abundant evidence that after in vivo traumatic brain injury, oxygen radicals contribute to changes in cerebrovascular structure and function; however, the cellular source of these oxygen radicals is not clear. The purpose of these experiments was to use a newly developed in vitro tissue culture model to elucidate the effect of strain, or stretch, on neuronal, glial, and endothelial cells and to determine the effect of the free radical scavenger polyethylene glycol-conjugated superoxide dismutase (PEG-SOD; pegorgotein, Dismutec) on the response of each cell type to trauma. METHODS Rat brain astrocytes, neuronal plus glial cells, and aortic endothelial cells were grown in cell culture wells with 2-mm-thick silastic membrane bottoms. A controllable, 50-millisecond pressure pulse was used to transiently deform the silastic membrane and thus stretch the cells. Injury was assessed by quantifying the number of cells that took up the normally cell-impermeable dye propidium iodide. Some cultures were pretreated with 100 to 300 U/mL PEG-SOD. RESULTS Increasing degrees of deformation produced increased cell injury in astrocytes, neuronal plus glial cultures, and aortic endothelial cells. By 24 hours after injury, all cultures showed evidence of repair as demonstrated by cells regaining their capacity to exclude propidium iodide. Compared with astrocytes or neuronal plus glial cultures, endothelial cells were much more resistant to stretch-induced injury and more quickly regained their capacity to exclude propidium iodide. PEG-SOD had no effect on the neuronal or glial response to injury but reduced immediate posttraumatic endothelial cell dye uptake by 51%. CONCLUSIONS These studies further document the utility of the model for studying cell injury and repair and further support the vascular endothelial cell as a site of free radical generation and radical-mediated injury. On the assumption that, like aortic endothelial cells, stretch-injured cerebral endothelial cells also produce oxygen radicals, our results further suggest the endothelial cell as a site of therapeutic action of free radical scavengers after traumatic brain injury.

Effect of Ca2+ on in vitro astrocyte injury

Abstract: Current literature suggests that a massive influx of Ca2+ into the cells of the CNS induces cell damage associated with traumatic brain injury (TBI). Using an in vitro model for stretch‐induced cell injury developed by our laboratory, we have investigated the role of extracellular Ca2+ in astrocyte injury. The degree of injury was assessed by measurement of propidium iodide uptake and release of lactate dehydrogenase. Based on results of in vivo models of TBI developed by others, our initial hypothesis was that decreasing extracellular Ca2+ would result in a reduction in astrocyte injury. Quite unexpectedly, our results indicate that decreasing extracellular Ca2+ to levels observed after in vivo TBI increased astrocyte injury. Elevating the extracellular Ca2+ content to twofold above physiological levels (2 mM) produced a reduction in cell injury. The reduction in injury afforded by Ca2+ could not be mimicked with Ba2+, Mn2+, Zn2+, or Mg2+, suggesting that a Ca2+‐specific mechanism is involved. Using 45Ca2+, we demonstrate that injury induces a rapid influx of extracellular Ca2+ into the astrocyte, achieving an elevation in total cell‐associated Ca2+ content two‐ to threefold above basal levels. Pharmacological elevation of intracellular Ca2+ levels with the Ca2+ ionophore A23187 or thapsigargin before injury dramatically reduced astrocyte injury. Our data suggest that, contrary to popular assumptions, an elevation of total cell‐associated Ca2+ reduces astrocyte injury produced by a traumatic insult.

S100B protein is released from rat neonatal neurons, astrocytes, and microglia by in vitro trauma and anti-S100 increases trauma-induced delayed neuronal injury and negates the protective effect of exogenous S100B on neurons.

S100B protein is found in brain, has been used as a marker for brain injury and is neurotrophic. Using a well‐characterized in vitro model of brain cell trauma, we have previously shown that strain injury causes S100B release from neonatal rat neuronal plus glial cultures and that exogenous S100B reduces delayed post‐traumatic neuronal damage even when given at 6 or 24 h post‐trauma. The purpose of the current studies was to measure post‐traumatic S100B release by specific brain cell types and to examine the effect of an antibody to S100 on post‐traumatic delayed (48 h) neuronal injury and the protective effect of exogenous S100B. Neonatal rat cortical cells grown on a deformable elastic membrane were subjected to a strain (stretch) injury produced by a 50 ms displacement of the membrane. S100B was measured with an ELISA kit. Trauma released S100B from pure cultures of astrocytes, microglia, and neurons. Anti‐S100 reduced released S100B to below detectable levels, increased delayed neuronal injury in traumatized cells and negated the protective effect of exogenous S100B on injured cells. Heat denatured anti‐S100 did not exacerbate injury. These studies provide further evidence for a protective role for S100B following neuronal trauma.

S100B protein is released by in vitro trauma and reduces delayed neuronal injury

S100B protein in brain is produced primarily by astrocytes, has been used as a marker for brain injury and has also been shown to be neurotrophic and neuroprotective. Using a well characterized in vitro model of brain cell trauma, we examined the potential role of exogenous S100B in preventing delayed neuronal injury. Neuronal plus glial cultures were grown on a deformable Silastic membrane and then subjected to strain (stretch) injury produced by a 50 ms displacement of the membrane. We have previously shown that this injury causes an immediate, but transient, nuclear uptake of the fluorescent dye propidium iodide by astrocytes and a 24–48 h delayed uptake by neurons. Strain injury caused immediate release of S100‐beta with further release by 24 and 48 h. Adding 10 or 100 nm S100B to injured cultures at 15 s, 6 h or 24 h after injury reduced delayed neuronal injury measured at 48 h. Exogenous S100B was present in the cultures through 48 h. These studies directly demonstrate the release and neuroprotective role of S100B after traumatic injury and that, unlike most receptor antagonists used for the treatment of trauma, S100B is neuroprotective when given at later, more therapeutically relevant time points.

Traumatic injury of cultured astrocytes alters inositol (1,4,5)–trisphosphate-mediated signaling

Our previous studies using an in vitro model of traumatic injury have shown that stretch injury of astrocytes causes a rapid elevation in intracellular free calcium ([Ca2+]i), which returns to near normal by 15 min postinjury. We have also shown that after injury astrocyte intracellular calcium stores are no longer able to release Ca2+ in response to signal transduction events mediated by the second messenger inositol (1,4,5)‐trisphosphate (IP3, Rzigalinski et al., 1998 ). Therefore, we tested the hypothesis that in vitro injury perturbs astrocyte IP3 levels. Astrocytes grown on Silastic membranes were labeled with [3H]–myo–inositol and stretch‐injured. Cells and media were acid‐extracted and inositol phosphates isolated using anion‐exchange columns. After injury, inositol polyphosphate (IPx) levels increased up to 10‐fold over uninjured controls. Significant injury‐induced increases were seen at 5, 15, and 30 min and at 24 and 48 h postinjury. Injury‐induced increases in IPx were equivalent to the maximal glutamate and trans‐(1S,3R)–1–amino–1,3–cyclopentanedicarboxylic acid‐stimulated IPx production, however injury‐induced increases in IPx were sustained through 24 and 48 h postinjury. Injury‐induced increases in IPx were attenuated by pretreatment with the phospholipase C inhibitors neomycin (100 μM) or U73122 (1.0 μM). Since we have previously shown that astrocyte [Ca2+]i returns to near basal levels by 15 min postinjury, the current results suggest that IP3‐mediated signaling is uncoupled from its target, the intracellular Ca2+ store. Uncoupling of IP3‐mediated signaling may contribute to the pathological alterations seen after traumatic brain injury. GLIA 33:12–23, 2001.

NMDA Receptor Activation Contributes to a Portion of the Decreased Mitochondrial Membrane Potential and Elevated Intracellular Free Calcium in Strain-Injured Neurons

In our previous studies, we have shown that in vitro biaxial strain (stretch) injury of neurons in neuronal plus glial cultures increases intracellular free calcium ([Ca(2+)](i)) and decreases mitochondrial membrane potential (deltapsi(m)). The goal of this study was to determine whether strain injury, without the addition of exogenous agents, causes glutamate release, and whether NMDA receptor antagonists affect the post-strain injury rise in [Ca(2+)](i) and decrease in deltapsi(m). [Ca(2+)](i) and deltapsi(m) were measured using the fluorescent indicators fura-2 AM and rhodamine-1,2,3 (rh123). Strain injury of neuronal plus glial cultures caused an immediate 100-200 nM elevation in neuronal [Ca(2+)]i and a decline in neuronal deltapsi(m) by 15 min post-injury. Pretreatment with the NMDA receptor antagonist MK-801 (10 microM) attenuated the [Ca(2+)](i) elevation after mild, but not moderate and severe injury. MK-801 pretreatment reduced the decline in deltapsi(m) after mild and moderate, but not after severe injury. The NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid (APV; 100 microM) had effects similar to MK-801. Simultaneous measurement of [Ca(2+)](i) and deltapsi(m) demonstrated a significant correlation and a temporal relationship between [Ca(2+)](i) elevation and depression of deltapsi(m). We conclude that NMDA receptor stimulation contributes to some of the changes in [Ca(2+)](i) and deltapsi(m) after less severe strain injury. However, after more pronounced injury other mechanisms appear to be more involved.