Decrease in reducing power of aqueous humor originating from glaucomatous rabbits.

AIM: To evaluate changes in the reducing power of aqueous humor (AH) with cyclic voltammetry (CV) and HPLC-EC. METHODS: NZW Rabbits exhibiting a sporadic mutation causing bilaterally buphthalmus eyes were set for intra ocular pressure (IOP) and eye size measurements. AH was obtained under anesthesia, from congenital glaucomatic rabbits (CGR, n=6) and age-matched controls (CON; n=6). The AH samples were analysed by CV and HPLC-EC. RESULTS: CGR IOP was found to be significantly higher than in CON (33.5+/-1.1 and 14.2+/-1.0 mmHg, respectively), eye size was 18.25 and 13.9 cm, respectively. CV analysis revealed two anodic currents representing two groups of low molecular weight antioxidant (LMWA). The two anodic potentials were equal for the two tested groups, indicating the same components of LMWA. The first anodic current of CGR was only 30% of the CON rabbits (2.11 vs7.17 microA/mg protein, t-test: P<0.05). As the main hydrophilic components of the first anodic current are known to be uric acid (UA) and ascorbic acid (AA), they were analysed for exact content by HPLC-EC. UA and AA levels were significantly lower in the CGR group (UA: 17.1+/-3.2 and 189.1+/-75.70 microM/mg, AA: 1.1+/-0.3 and 4.8+/-2.0 microM/mg protein respectively). CONCLUSIONS: Changes in the reducing power, as indicated by CV analysis, of CGR AH, is probably a result of chronic oxidative stress caused by the pathology. The differences in the first anodic wave are mainly due to a fall in the concentration of UA and AA.

Induction of antitumor immunity by indomethacin.

Irradiated tumor cells given, together with indomethacin, to syngeneic mice induced an antitumor response and conferred protection against a challenge of a lethal dose of murine mammary (4T1) and lung (3LL) carcinoma cells. Continuous administration of indomethacin was crucial throughout the entire period of immunization and challenge, as no protection was achieved when the drug was given during only one of these procedures. Antitumor immunity was long-lasting and, when tested in the 4T1 model, 48% of mice were resistant to a second challenge of lethal tumor cells. Tumor-free immune mice that were given indomethacin for more than 300 days remained healthy with normal white blood cell counts and normal spleen size. Cells isolated from immune mice were able to kill tumor cells in culture after in vitro activation by interleukin-2, in a manner similar to cells from naive normal control mice. In addition, the mitogenic response of their T cells was as high as that of the control naive mice. While indomethacin was able to induce antitumor immunity to 4T1 and 3LL murine carcinoma cells, both of which contain a high concentration of endogenic prostaglandin E(2) (PGE2), no such immunity was achieved to murine tumor cells with a low concentration of endogenic PGE2. These results suggest a correlation between PGE2 concentration and the ability of indomethacin to induce antitumor immunity. We therefore suggest that an immunotherapy protocol with long-term dispensation of a tolerable dose of an immunomodulator, given together with irradiated autologous tumor cells, may stimulate antitumor responses to tumors containing high concentrations of endogenic PGE2.

Rasagiline, a monoamine oxidase-B inhibitor, protects NGF-differentiated PC12 cells against oxygen-glucose deprivation.

In our in vitro model, rasagiline a selective irreversible monoamine oxidase-B (MAO-B) inhibitor, protected nerve growth factor (NGF)-differentiated PC12 cells from cell death under oxygen and glucose deprivation (OGD). The severity of the OGD insult, as expressed by cell death, was time-dependent. Exposure of the cells to OGD for 3 hr followed by 18 hr of reoxygenation caused about 30-40% cell death. Under these conditions, the neuroprotective effect of rasagiline was dose-dependent: rasagiline reducing OGD-induced cell death by 68% and 80% at 100 nM and 1 microM, respectively. The neuroprotective effect of rasagiline was also observed when added after the OGD insult (55% reduction in cell death). Under rasagiline treatment, there was a lesser decrease in ATP content in cultures exposed to OGD compared with that in untreated cultures. OGD followed by reoxygenation resulted in a several fold increase in PGE(2) release into the extracellular medium. Rasagiline (100 nM-1 microM) markedly inhibited OGD-induced PGE(2) release. Clorgyline, a monoamine oxidase-A (MAO-A) inhibitor, did not protect NGF-differentiated PC12 cells against OGD-induced cell death. As NGF-differentiated PC12 cells contain exclusively MAO type A, these data suggest that the neuroprotective effect of rasagiline under OGD conditions is independent of MAO inhibition.

Antioxidants attenuate acute toxicity of tumor necrosis factor-alpha induced by brain injury in rat.

Tumor necrosis factor-alpha alpha (TNF-alpha) and reactive oxygen species (ROS) are produced in the brain after traumatic injury and have deleterious effects. In a rat model of closed head injury (CHI), the synthetic antioxidant from the nitroxide family, Tempol, improved recovery and protected the blood-brain barrier. Similar protection was found after CHI in heat-acclimated rats, in which the endogenous antioxidants have been shown to be elevated after CHI. The present study examined the relationship between TNF-alpha and ROS after CHI, namely, whether after CHI, antioxidants that afforded cerebroprotection also attenuated brain levels of TNF-alpha. Three groups of rats were subjected to CHI: (1) control, nontreated, (2) Tempol-treated, and (3) heat-acclimated (30 days at 34 degrees C). Four hours after injury (time for peak production of TNF-alpha), the activity of TNF-alpha was measured. Although clinical recovery was facilitated in rats of the two treated groups, TNF-alpha activity was as high as in the traumatized, untreated rats. Moreover, direct injection of TNF-alpha into mouse brain induced disruption of the blood-brain barrier, indicating its acute harmful effect. This toxic effect was attenuated by before and after treatment with Tempol. Our results support the hypothesis that in vivo antioxidants neutralize TNF-alpha toxicity, probably by interfering with activation of the transcription factor NF-kappa-B.

Closed head injury in the rat induces whole body oxidative stress: overall reducing antioxidant profile.

Traumatic injury to the brain triggers the accumulation of harmful mediators, including highly toxic reactive oxygen species (ROS). Endogenous defense mechanism against ROS is provided by low molecular weight antioxidants (LMWA), reflected in the reducing power of the tissue, which can be measured by cyclic voltammetry (CV). CV records biological peak potential (type of scavenger), and anodic current intensity (scavenger concentration). The effect of closed head injury (CHI) on the reducing power of various organs was studied. Water and lipid soluble extracts were prepared from the brain, heart, lung, kidney, intestine, skin, and liver of control and traumatized rats (1 and 24 h after injury) and total LMWA was determined. Ascorbic acid, uric acid, alpha-tocopherol, carotene and ubiquinol-10 were also identified by HPLC. The dynamic changes in LMWA levels indicate that the whole body responds to CHI. For example, transient reduction in LMWA (p<0.01) in the heart, kidney, lung and liver at 1 h suggests their consumption, probably due to interaction with locally produced ROS. However, in some tissues (e.g., skin) there was an increase (p<0.01), arguing for recruitment of higher than normal levels of LMWA to neutralize the ROS. alpha-Tocopherol levels in the brain, liver, lung, skin, and kidney were significantly reduced (p<0.01) even up to 24 h. We conclude that although the injury was delivered over the left cerebral hemisphere, the whole body appeared to be under oxidative stress, within 24 h after brain injury.

Antioxidant mechanisms in apolipoprotein E deficient mice prior to and following closed head injury.

Apolipoprotein E deficient mice have distinct memory deficits and neurochemical derangements and their recovery from closed head injury is impaired. In the present study, we examined the possibility that the neuronal derangements of apolipoprotein E deficient mice are associated with oxidative stress, which in turn affects their ability to recover from close head injury. It was found that brain phospholipid levels in apolipoprotein E deficient mice are lower than those of the controls (55+/-15% of control, P<0. 01), that the cholesterol levels of the two mice groups are similar and that the levels of conjugated dienes of the apolipoprotein E deficient mice are higher than those of control mice (132+/-15% of P<0.01). Brains of apolipoprotein E deficient mice had higher Mn-superoxide dismutase (134+/-7%), catalase (122+/-8%) and glutathione reductase (167+/-7%) activities than control (P<0.01), whereas glutathione peroxidase activity and the levels of reduced glutathione and ascorbic acid were similar in the two mouse groups. Closed head injury increased catalase and glutathione peroxidase activities in both mouse groups, whereas glutathione reductase increased only in control mice. The superoxide dismutase activity was unaffected in both groups. These findings suggest that the antioxidative metabolism of apolipoprotein E deficient mice is altered both prior to and following head injury and that antioxidative mechanisms may play a role in mediating the neuronal maintenance and repair derangements of the apolipoprotein E deficient mice.

Characterization of pardaxin-induced dopamine release from pheochromocytoma cells: role of calcium and eicosanoids.

Pardaxin, an excitatory neurotoxin, induced dopamine release from pheochromocytoma (PC12) cells both in the presence and absence of extracellular calcium ([Ca]o). In the presence of extracellular calcium, nifedipine, an L-type calcium channel blocker, did not affect dopamine release, whereas 1,2-bis (2-aminophenoxy) ethane N,N, N'N'-tetra-acetic acid (BAPTA), a chelator of cytosolic calcium, and dantrolene, a blocker of calcium release from intracellular stores, inhibited only partially (30-40%) pardaxin-induced dopamine release. In the absence of [Ca]o, BAPTA and dantrolene were ineffective. Pardaxin stimulated the arachidonic acid (AA) cascade in PC12 cells independently of [Ca]o. The phospholipase inhibitors mepacrine and bromophenacyl bromide inhibited both pardaxin-induced AA release and pardaxin-induced dopamine release. Dopamine release induced by pardaxin also was blocked by the lipoxygenase inhibitors nordihydroguaiaretic acid, esculetin, and 2-(12-hydroxydodeca-5, 10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone. Under these conditions, a parallel reduction in 5-hydroxyeicosatetranoic acid release also was observed. Suppression of pardaxin-induced dopamine release by inhibitors of phospholipase A2 and lipoxygenase was more pronounced in calcium-free medium. These results indicate the involvement of the lipoxygenase pathway in pardaxin-induced dopamine release and suggest the use of this toxin as a novel pharmacological tool for investigating the mechanism of calcium-independent neurotransmitter release.

Pardaxin, a new pharmacological tool to stimulate the arachidonic acid cascade in PC12 cells.

The effect of Pardaxin, a neurotoxin that induces neurotransmitter release from neurons, on the arachidonic acid (AA) cascade was studied in PC12 cells. Both native and the synthetic Pardaxin selectively stimulated phospholipase A2 (PLA2) activity (measured by [3H]AA release) in the presence as well as in the absence of extracellular calcium. Pardaxin-stimulated PLA2 activity was also evident in the increased formation of lysophosphatidylcholine. Pardaxin analogs, lacking the alpha-helical structure that is essential for insertion into the plasma membrane, were ineffective in stimulating the AA cascade in PC12 cells. Pardaxin stimulation of PLA2 was markedly inhibited by the nonselective PLA2 inhibitors bromophenacyl bromide and mepacrine, by methyl arachidonyl fluorophosphonate, a dual inhibitor of calcium-dependent cytosolic PLA2 and the calcium-independent PLA2 and by bromoenol lactone[(E)-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl-2H-pyran -2- one], a highly specific inhibitor of calcium-independent PLA2. After Pardaxin treatment, there was increased release of AA metabolites produced by the cyclooxygenase pathway as expressed in an 8-fold increase of PGE2 release. The release of other eicosanoids, such as 6-keto-PGF1alpha and thromboxane B2, was also augmented. Pardaxin-induced PGE2 release was observed in calcium-free medium and in the absence of any increase in cytosolic calcium. Dexamethasone partially inhibited Pardaxin-induced PGE2 release. This effect was reversed by the type II corticosteroid receptor antagonist RU-38486. Our results indicate that Pardaxin stimulates release of AA and eicosanoids, independently of calcium, and suggest that calcium-independent PLA2 plays an important role in Pardaxin stimulation of the AA cascade.

Neuroprotection against oxidative stress by serum from heat acclimated rats.

Exposure of PC12 cells, to 1% serum derived from normothermic (CON) rats resulted in 79% cell death. Sister cultures treated with 1% serum derived from heat acclimated (ACC) rats, were neuroprotected and expressed a significant reduction in cell death. In PC12 cells exposed to a free radical generator causing an oxidative stress, 90% cell death was measured in CON serum treated cultures, while ACC serum treated cultures were neuroprotected. Xanthine oxidase activity and uric acid (UA) levels were lower in ACC serum compared to CON. Addition of UA to both sera abolished the difference in cell viability, and toxicity of ACC serum reached that of CON. These findings suggest a causal relationship between the lower levels of UA in ACC and the neuroprotective effect observed. The present study proposes heat acclimation as an experimental and/or clinical tool for the achievement of neuroprotection.

Mechanism of brain protection by nitroxide radicals in experimental model of closed-head injury.

Reactive oxygen-derived species were previously implicated in mediation of post-traumatic brain damage; however, the efficacy of traditional antioxidants in preventing/reversing the damage is sometimes limited. The present work focused on the mechanisms underlying the neuroprotective activity of cell permeable, nontoxic, antioxidants, namely stable nitroxide radicals in an experimental model of rat closed-head injury. Brain damage was induced by the weight-drop method and the clinical status was evaluated according to a neurological severity score at 1 h and 24 h, where the difference between these scores reflects the extent of recovery. The metal chelator deferoxamine as well as three nitroxide derivatives, differing in hydrophilicity and charge, and one hydroxylamine (a reduced nitroxide) facilitated the clinical recovery and decreased the brain edema. The nitroxides, but neither the hydroxylamine nor deferoxamine, protected the integrity of the blood-brain barrier. Superoxide dismutase also improved the clinical recovery but did not affect brain edema or the blood-brain barrier. The results suggest that by switching back and forth between themselves, the nitroxide and hydroxylamine act catalytically as self-replenishing antioxidants, and protect brain tissue by terminating radical-chain reactions, oxidizing deleterious metal ions, and by removal of intracellular superoxide.

Reduced levels of antioxidants in brains of apolipoprotein E-deficient mice following closed head injury.

Recent animal model studies using apolipoprotein E (apoE)-deficient (knockout) mice revealed that these mice have memory deficits and neurochemical derangements and that they recover from closed head injury less adequately than control mice. In the present study, we examined the possibility that the diminished recovery of apoE-deficient mice from head injury is related to a reduction in their ability to counteract oxidative damage. Measurements of reducing agents by cyclic voltammetry revealed that cortical homogenates of apoE-deficient and control mice contain similar levels of these compounds whose oxidation potentials for the two groups of mice are at 400 +/- 40 mV and 900 +/- 50 mV. The responses of the apoE-deficient and control groups to closed head injury were both biphasic and were composed of initial reductions followed by subsequent increases in the levels of reducing antioxidant equivalents. However, the two groups differed markedly in the magnitude of their response. This difference was most pronounced with the 400-mV reducing compounds, such that at 4 h after injury their levels in injured control mice increased over twofold relative to the noninjured control mice, whereas the corresponding anodic current of the apoE-deficient mice recovered only to its original level and did not increase further even by 24 h after injury. In vitro studies using recombinant apoE allele E3 and beta very low density lipoprotein revealed that this lipoprotein can delay Cu(2+)-induced lipid peroxidation. This suggests that the inability of the apoE-deficient mice to respond to brain injury by a surge in brain reducing compounds may be related, at least in part to direct antioxidant activity of apoE.

Changes of biological reducing activity in rat brain following closed head injury: a cyclic voltammetry study in normal and heat-acclimated rats.

Reactive oxygen species (ROS) are normally generated in the brain during metabolism, and their production is enhanced by various insults. Low molecular weight antioxidants (LMWA) are one of the defense mechanisms of the living cell against ROS. The reducing capacity of brain tissue (total LMWA) was measured by cyclic voltammetry (CV), which records biological oxidation potential specific to the type of scavenger(s) present and anodic current intensity (Ia), which depends on scavenger concentration. In the present study, the reducing capacity of rat brain following closed head injury (CHI) was measured. In addition, CV of heat-acclimated traumatized rats was used to correlate endogenous cerebroprotection after CHI with LMWA activity. Sham-injured rat brains displayed two anodic potentials: at 350 +/- 50 mV (Ia = 0.75 +/- 0.06 microA/mg protein) and at 750 +/- 50 mV (Ia = 1.00 +/- 0.05 microA/mg protein). Following CHI, the anodic waves appeared at the same potentials as in the sham animals. However, within 5 min of CHI, the total reducing capacity was transiently decreased by 40% (p < 0.01). A second dip was detected at 24 h (60%, p < 0.005). By 48 h and at 7 days, the Ia levels normalized. The acclimated rats displayed anodic potentials identical to those of normothermic rats. However, the Ia of both potentials was lower (60% of control, p < 0.001). The Ia profile after CHI was the direct opposite of the normothermic Ia profile: no immediate decrease of Ia and an increase from 4 h and up to 7 days (40-50%, p < 0.001). We suggest that the lowered levels of LMWA in the post-CHI period reflect their consumption due to overproduction of free radicals. The augmented concentration of LMWA found in the brain of the heat-acclimated rats suggests that these rats are better able to cope with these harmful radicals, resulting in a more favorable outcome following CHI.

An experimental model of closed head injury in mice: pathophysiology, histopathology, and cognitive deficits.

The present study describes the characterization of an experimental model of closed head injury (CHI) in the mouse. This model is a modification of a setup described and used previously in the rat. The weight-drop device was modified and adapted to the size and weight of the mouse and the typical parameters that define the severity of the injury and its outcome were evaluated. The posttraumatic accumulation of water, i.e., cerebral edema, the disruption of the blood-brain barrier (BBB), histopathology, motor and cognitive functions were studied up to 30 days following CHI. Increases in cerebral water content and of BBB permeability were observed in the injured hemisphere at 4 h (p < 0.05) and 24 h (p < 0.01) postinjury, respectively. By 7 days, edema disappeared, while the BBB remained open for up to 30 days. The motor function was evaluated by a set of criteria termed neurological severity score (NSS). NSS was severely impaired immediately after CHI and later showed a spontaneous progressive recovery, although some residual deficits, mainly of beam-walk and balance, were still present at 30 days. Mice trained in the Morris water maze before the injury demonstrated highly significant deficits in memory retention up to at least 11 days postinjury (p < 0.01). Histopathological analysis revealed significant neuronal cell death in CA1, CA2, and CA3 regions of the left hippocampus following CHI. However, in the right hippocampus, overt neuronal cell death was observed only in area CA3 at 7 days after CHI. These results suggest that the modified model of CHI in mice can reproduce the posttraumatic sequelae observed in rats and show that some of the data obtained in this model are essentially similar to those observed in human head injury. The experimental model of CHI in mice may be a useful tool for studies in animals that carry specific genetic alterations, aimed at manipulating neurochemical pathways involved in the pathophysiology of brain damage.

Cerebroprotective effect of stable nitroxide radicals in closed head injury in the rat.

Nitroxide stable radicals are unreactive toward most diamagnetic molecules, but readily undergo one-electron redox reactions with paramagnetic species such as free radicals and transition metals, thus serving as cell permeable antioxidants. The involvement of reactive oxygen species in the pathophysiology of neurotrauma has been well established. The neuroprotective properties of three nitroxides: 2,2,6,6-tetramethylpiperidine-1-N-oxyl (TPO), the hydrophilic analog: TPL, and its reduced form: TPH, were tested in a rat model of closed head injury (CHI). CHI was induced in ether anesthetized rats by a weight drop device and recovery was followed for up to 24 h. The "clinical status' was evaluated according to a "Neurological Severity Score' (NSS), at 1 h and 24 h, the difference between these scores, delta NSS, reflecting the extent of recovery. Edema was assessed by measurement of water content at 24 h. The integrity of the blood-brain barrier (BBB) was investigated using Evans Blue extravasation. TPL, TPH and TPO facilitated clinical recovery, the latter causing a more pronounced effect (delta NSS = 7.63 +/- 0.26 in treated rats vs 4.94 +/- 0.48 in control rats, P < 0.001). TPL was found to significantly reduce edema formation (80.13% +/- 0.26 vs 83.65% +/- 0.49, P < 0.001) and to ameliorate BBB disruption (P < 0.001). The therapeutic window of TPL was found to be in the range of 4 h after CHI. The mechanisms underlying the nitroxide neuroprotective activity presumably involve: (a) reoxidation of reduced transition metal ions; (b) a selective radical-radical reaction; and (c) catalytic removal of intracellular and extracellular .O2-. The results indicate that nitroxides could be used in neuroprotective treatment of CHI.

A novel nonpsychotropic cannabinoid, HU-211, in the treatment of experimental pneumococcal meningitis.

Typical features of pneumococcal meningitis have been demonstrated in rats inoculated with Streptococcus pneumoniae. HU-211, a novel noncompetitive N-methyl-D-aspartate antagonist recently demonstrated to inhibit tumor necrosis factor-alpha production under various conditions, improves recovery in some experimental models of brain injury. The present study tested the efficacy of HU-211 in combination with antimicrobial therapy in reducing brain damage in experimental pneumococcal meningitis. S. pneumoniae-infected rats were treated with saline alone, ceftriaxone alone, or with combination of ceftriaxone and HU-211 18 h after inoculation of the bacteria. Brain edema and blood-brain barrier impairment 48 h after infection were significantly (P<.05) reduced suggest that HU-211 when given concomitantly with antibiotics attenuates brain damage in the rat model of pneumococcal meningitis.

Polyamines induce blood-brain barrier disruption and edema formation in the rat.

Polyamines (PA) are derived from ornithine by the enzyme ornithine decarboxylase (ODC), which is activated very rapidly as acute and delayed responses to brain ischemia and trauma. Polyamines play a role in the disruption of the blood-brain barrier (BBB) in different pathological states. This study examined the effect of exogenous polyamines, administered intracerebrally (i.c.v.) or intracarotidly on BBB function. Putrescine, spermidine and spermine, given individually, were found to disrupt BBB integrity within 15 min of i.c.v. administration (p = 0.03; p = 0.0013; p = 0.042 vs saline treated rats, respectively). The effect was still evident after 1 h; however, since the saline treated rats also showed increased permeability of Evans blue at this time, there was no statistical difference between polyamines or saline treated rats 1 h post injection. When injected into the carotid artery, rapid increase in BBB permeability was found 1 min after putrescine and spermidine (p < 0.01 vs saline), with a slight decline at 15 min. A slower effect was noticed after spermine administration which reached significance only at 15 min. These results suggest a role for PA as mediators of vasogenic edema formation in the brain soon after brain injuries which induce increased production of these compounds.

The effect of the adrenocortical axis upon recovery from closed head injury.

Fragments and analogs of the hormone ACTH were previously shown to have beneficial effect on the outcome of head injury, while elevated levels of corticosterone (CS) exacerbate it. In the present study we investigated the role of the hypothalamo-pituitary-adrenal (HPA) axis in the pathophysiology of closed head injury (CHI). CHI was produced in ether-anesthetized rats by a calibrated weight-drop device. After evaluating the functional status according to a set of criteria, at 1 and 24 h, the rats were sacrificed and cortical tissue was removed to determine its water content. CHI was also produced in rats that underwent surgical procedures to remove their adrenal gland (ADEX) or the pituitary (HypoX), thus altering the levels of their circulating HPA hormones. Given after CHI, to rats with intact HPA axis, ACTH reduced edema and improved recovery. ADEX rats (6 days postsurgery) had 10-fold higher levels of plasma ACTH. ADEX rats subjected to CHI showed improved functional outcome (p = 0.008) and reduced edema (p = 0.02). We then produced CHI in three groups of rats: HypoX (15 days postsurgery), HypoX treated with ACTH, and controls. In HypoX rats, CHI resulted in increased mortality (35% vs 0) and edema in the surviving rats, and a slower recovery, as compared with the control. Mortality was prevented, edema slightly reduced, and recovery significantly improved after administration of 1-24-ACTH to HypoX rats with CHI. Our results suggest that ACTH has a cerebroprotective effect on the outcome of CHI.

High oligomycin concentrations augment 6-keto-PGF1 alpha production in ventricular cardiomyocytes.

Incubation of cultured ventricular cardiomyocytes with high oligomycin concentrations (100 micrograms/ml), either alone or combined with 2-deoxyglucose (20 mM), led to the rapid depletion of cellular ATP. Inositol (poly)phosphate production decreased, and 6-keto PGF1 alpha production was increased. In cells depleted of ATP, either by low oligomycin concentrations or by sodium azide, 6-keto PGF1 alpha was not appreciably increased. There was a 25% rise in the release of fatty acids from the sn-2 position in glycerophospholipids. We suggest that oligomycin at high concentrations causes the release of free arachidonic acid from phospholipids either by non-PIP2-specific PLC and DG lipase or by phospholipase D, phosphatidic acid phosphatase and DG lipase. The effect is unrelated to decreased cellular ATP content.

A tissue culture ischemic device to study eicosanoid release by pheochromocytoma PC12 cultures.

In an attempt to search for neuronal models to investigate the molecular pharmacology of central nervous system ischemia, we have focused on PC12 pheochromocytoma cultures which are now popular in neuroscience research. These chromaffinergic transformed cells, originary from the adrenal medulla, synthesize and release catecholamines and, upon treatment with nerve growth factor (NGF), differentiate to a sympathetic phenotype expressing neurites and excitability. To measure eicosanoid production, undifferentiated or NGF-treated PC12 cultures have been exposed for 1 h to a mixture of N2/CO2 (95:5%), resulting in hypoxia (5 +/- 1% O2), followed by 1 h reoxygenation (21% O2) using a special ischemic device. Hypoxia, up to 2 h, was not followed by significant cytotoxicity or significant production of prostaglandin PGE2. However, upon reoxygenation, a specific release of PGE2 (2-3 fold over control) was measured. A similar PGE2-enhanced release could be induced by 'chemical hypoxia' using 2-deoxyglucose and oligomycin to reduce cellular adenosine triphosphate (ATP). Anoxia (0.1-1% O2, 1 h) achieved by a reduction of culture incubation volume and the reduction in ATP level have been found as critical parameters leading to PC12 cells cytotoxicity. These results emphasize the simplicity and applicability of the tissue culture ischemic device proposed to investigate hypoxia and ischemia at a cellular level.

Cytolysins increase intracellular calcium and induce eicosanoids release by pheochromocytoma PC12 cell cultures.

Cytolysins are the most commonly occurring toxins among bacteria, plants, and animals. By distributing cell membrane, they impair ionic permeability, leading to cell death. In an attempt to investigate cytolysin action on catecholaminergic neurons, we have treated pheochromocytoma cell cultures with Streptolysin S, Staphylococcus aureus alpha and delta, Stoichatus, Parcelsin, and cobra direct lytic factor. To measure neurotoxicity, PC12 cultures were loaded with 51Cr and exposed for 1 hr at 37 degrees C to different concentrations of cytolysins. Cytotoxic dose-response curves have been generated resulting in CD50 (cytotoxic dose 50%) in the range of 1-50 micrograms toxin/culture. Using subcytotoxic concentrations of cytolysins (which are of clinical relevance), changes on intracellular calcium were measured by Fura-2 fluorescence technique. Addition of either Stoichatus toxin and tetanolysin or streptococcus and staphylococcus cytolysins to PC12 cells caused rapidly or gradually a progressive increase in [Ca2+]i, respectively. Under similar conditions, samples of PC12 culture medium were assayed for 3H-arachidonic acid released and by radioimmunoassay for the content of PGE2 (prostaglandin), TXB2 (stable metabolite of thromboxane), and 5-HETE (hydroxy acid lipoxygenase product). PLA2 was activated 4.5-6.0-fold and the levels of all three eicosanoids were increased by 2.5-9-fold (PGE2), 4-6-fold (TXB2), and over 100-fold (5-HETE) by Stoichatus and Parcelsin cytolysins. Upon treatment with Streptolysin S and staphylococcus delta toxins PLA2 (phospholipase A2) was slightly activated (1.5-fold) and the levels of PGE2 and TXB2 increased 1.3-2.0-fold and that of 5-HETE up to 30-fold.(ABSTRACT TRUNCATED AT 250 WORDS)