Beverly A. Rzigalinski

Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock

Anandamide, an endogenous cannabinoid ligand, binds to CB1 cannabinoid receptors in the brain and mimics the neurobehavioural actions of marijuana,. Cannabinoids and anandamide also elicit hypotension mediated by peripheral CB1 receptors. Here we report that a selective CB1 receptor antagonist, SR141716A, elicits an increase in blood pressure in rats subjected to haemorrhagic shock, whereas similar treatment of normotensive rats or intracerebroventricular administration of the antagonist during shock do not affect blood pressure. Blood from haemorrhaged rats causes hypotension in normal rats, which can be prevented by SR141716A but not by inhibition of nitric oxide synthase in the recipient. Macrophages and platelets from haemorrhaged rats elicit CB1 receptor-mediated hypotension in normotensive recipients, and incorporate arachidonic acid or ethanolamine into a product that co-elutes with anandamide on reverse-phase high-performance liquid chromatography. Also, macrophages from control rats stimulated with ionomycin or bacterial phospholipase D produce anandamide, as identified by gas chromatography and mass spectrometry. These findings indicate that activation of peripheral CB1 cannabinoid receptors contributes to haemorrhagic hypotension, and anandamide produced by macrophages may be a mediator of this effect.

Reduction of Voltage-Dependent Mg2+ Blockade of NMDA Current in Mechanically Injured Neurons

Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury. Here, the effects of mechanical injury on the voltage-dependent magnesium (Mg2+) block of NMDA currents in cultured rat cortical neurons were examined. Stretch-induced injury was found to reduce the Mg2+ blockade, resulting in significantly larger ionic currents and increases in intracellular free calcium (Ca2+) concentration after NMDA stimulation of injured neurons. The Mg2+ blockade was partially restored by increased extracellular Mg2+ concentration or by pretreatment with the protein kinase C inhibitor calphostin C. These findings could account for the secondary pathological changes associated with traumatic brain injury.

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.

Alterations in Phosphatidylcholine Metabolism of Stretch‐Injured Cultured Rat Astrocytes

Abstract: The primary objective of this study was to determine the influence of stretch‐induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silastic‐bottomed tissue culture wells in medium that was usually supplemented with 10 µM unlabeled arachidonate. Cell injury was produced by stretching (5–10 mm) the Silastic membrane with a 50‐ms pulse of compressed air. Stretch‐induced cell injury increased the incorporation of [3H]choline into PC in an incubation time‐ and stretch magnitude‐dependent manner. PC biosynthesis was increased three‐ to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch‐induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A2 (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch‐induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretrating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch‐dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n‐acetylcysteine). Arachidonate‐enriched cells were more susceptible to stretch‐induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non‐arachidonate‐enriched cells. In summary, the data suggest that stretch‐induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron‐catalyzed Haber‐Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cells membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch‐induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch‐induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction.

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.

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.

Antagonism of group I metabotropic glutamate receptors and PLC attenuates increases in inositol trisphosphate and reduces reactive gliosis in strain-injured astrocytes.

We have previously found that in vitro traumatic injury uncouples IP3-mediated intracellular free calcium ([Ca2+]i) signaling in astrocytes (Rzigalinski et al., 1998; Floyd et al., 2001). Since Group I metabotropic glutamate receptors (mGluRs) are coupled to IP3-mediated Ca2+ signaling, we investigated their role in the in vitro strain injury of cultured astrocytes. Astrocytes grown on Silastic membranes were labeled with 3H-myo-inositol and strain (stretch)-injured. Cells injured in the presence of LiCl to prevent inositol phosphate metabolism were acid extracted and inositol phosphates (IPx) isolated using anion exchange columns. Reactive gliosis was assessed as increased glial fibrillary acidic protein immunoreactivity (GFAP-IR). Pre- but not post-injury administration of (RS)-1-aminoindan-15-decarboxylic acid (AIDA) or (S)-4-carboxy-3-hydroxyphenylglycine (S4CH3HPG), both group I mGluR antagonists, attenuated injury-induced increases in IPx. Injury increased GFAP-IR in astrocytes at 24 and 48 h post injury, which was reduced or blocked by AIDA or inhibition of phospholipase C (PLC) with U73122. These findings suggest that strain injury activates Group I mGluRs, causing aberrant IPx production and uncoupling of the PLC signaling pathway. Changes in this signaling pathway may be related to induction of reactive gliosis. Additionally, our results suggest a complex physical coupling between G protein receptor, PLC, and IP3 receptor, in support of the conformational coupling model.