Stephen M. Taylor

Virus-induced enhancement of arachidonate metabolism by bovine alveolar macrophages in vitro.

Virus infection of alveolar macrophages both in vivo and in vitro has been associated with a variety of changes in cellular function. Some of these changes are identical to the effects that arachidonate‐derived mediators, prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids, have on macrophage function. Virus infection of macrophages has been previously shown to increase the output of some arachidonate metabolites, most notably PGE2. However, the effect of virus infection on arachidonate metabolism in general has not been well described. In our experiments, primary cultures of alveolar macrophages obtained from normal cattle by bronchoalveolar lavage, were infected in vitro with parainfluenza type 3 virus. At days 0 to 4 post‐infection (p.i.) these cells were labelled with 3H‐arachidonic acid and stimulated with either serum‐coated zymosan, the calcium ionophore A23187, or phorbol myristate acetate. The complete spectrum of arachidonate‐derived metabolites was determined by reverse‐phase high performance liquid chromatography with UV and on‐line radiometric monitoring of column eluant. The total output of metabolites of arachidonic acid by virus‐infected alveolar macrophages was increased over that of noninfected controls (with all stimuli tested) by day 4 p.i. (P ≤ 0.05). The production of metabolites by the cyclooxygenase, 12‐and 5‐lipoxygenase enzyme systems was significantly increased, as was the release of 3H‐arachidonate. The lack of stimulus specificity and the increases in arachidonate release suggest that greater substrate availability, due either to increased phosphollpase activity or direct virus‐membrane interaction, may be responsible for the virus‐induced enhancement of metabolite output.

Decreased Muscarinic Acetylcholine Receptor Number in the Central Nervous System of the Tottering (tg/tg) Mouse

Abstract: The tottering mouse (tg/tg) is a single‐locusmutant, phenotypically characterized by the development of epilepsy associated with distinct electroencephalo‐graphic abnormalities. Because of reported alterations in muscarinic receptor (mAChR) number in various seizure states, mAChR density was examined in discrete brain regions of tottering (tgltg) and coisogenic wild‐type (+/+) mice. Saturation binding experiments revealed a widespread decrease in membrane mAChR density in the CNS of adult tottering (tg/tg) mice as compared with age‐matched control wild‐type (+/+) mice. The decrease was most pronounced in the hippocampus, where tg/tg mice exhibited a 40‐60% reduction in mAChR density with no change in the affinity of the receptor for antagonists or agonists. At postnatal day 10, before the reported onset of electroencephalographic abnormalities, 114 and 65% increases in mAChR density were observed in the tg/tg hippocampus and cortex, respectively. Following the development of seizure activity at postnatal day 22, mAChR density in the tg/tg hippocampus was reduced by 29%. No change in brain mAChR density was seen in adult heterozygotes (+/tg), which do not develop electroencephalographic or seizure abnormalities. These results indicate that the development of reduced mAChR number in the CNS of the tg/tg mouse is secondary to abnormal neuronal activity, providing further support for the hypothesis that membrane depolarization can cause a decrease in neuronal mAChR density.

Reversal of virus-induced alveolar macrophage bactericidal dysfunction by cyclooxygenase inhibition in vitro.

Virus infection of alveolar macrophages (AM) both in vivo and in vitro has been associated with a decreased ability of these cells to kill bacteria, together with enhanced production of metabolites of arachidonic acid. These metabolites, especially PGE2, may be inhibitory to some phagocyte functions. Primary cultures of bovine AM obtained by bronchoalveolar lavage of normal cattle were infected in vitro with parainfluenza‐3 (PI3 virus) virus. Killing of Staphylococcus epidermidis by AM was determined on days 1‐4 post‐infection (p.i.) PI3 virus‐infected AM killed significantly fewer bacteria on day 4 p.i. compared to uninfected controls (12.1 ± 1.3% infected vs. 52.7 ± 7.2% controls, P ≤ 0.05). Bacterial killing by virus‐infected AM, but not control AM, was significantly enhanced on day 4 p.i. by addition of cyclooxygenase inhibitors 1 hr prior to bactericidal assay (28.0 ± 4.5% indomethacin, 36.0 ± 4.1% mefenamic acid, 38.6 ± 7.3% piroxicam, 37.0 ± 6.4% NDGA, 44.9 ± 7.7% ETYA, P ≤ 0.05). Phagocytosis of opsonized sheep erythrocytes and superoxide generation by virus‐infected AM were not significantly increased by cyclooxygenase inhibition. Phagosome‐lysosome fusion was severely impaired in virus‐infected AM. Pretreatment of virus‐infected AM with indomethacin significantly enhanced the percentage of cell expressing fusion activity. This data suggests that in vitro bactericidal dysfunction associated with virus infection of AM is partially the result of enhanced production of prostaglandins or thromboxane by AM and/or an abnormal response to normal levels of endogenously produced cyclooxygenase metabolites. The data further indicate the presence of cyclooxygenase sensitive (phagosome‐lysosome fusion) and insensitive (phagocytic) components of virus‐induced bactericidal dysfunction in AM.

Stimulation of arachidonic acid metabolism in silica-exposed alveolar macrophages

The molecular events involved in both the initiation and development of silicosis are at present poorly defined, although mediators released from macrophages exposed to silica particles are believed to play a role. We have investigated the in vitro production of arachidonic acid (AA) metabolites in adherent bovine alveolar macrophages (BAM) incubated with crystalline silica. BAM were prelabeled with 3H-AA and incubated with 0.5-5.0 mg silica. Lipid metabolites released into the culture medium were analyzed by high-performance liquid chromatography. Simultaneously, lactate dehydrogenase (LDH) was assayed to provide an indication of cell injury. No 5-lipoxygenase metabolites were detected at the lowest silica dose tested (0.5 mg/well), but 5-hydroxyeicosatetraenoic acid (5-HETE) was the major AA metabolite detected between 1.5 and 5.0 mg of silica. A fivefold increase in the production of leukotriene B4 (LTB4) and its two nonenzymatic diastereomers (Isomers I and II) was observed as the silica concentration was increased from 1.0 to 5.0 mg. In contrast, the release of cyclooxygenase products declined with increasing concentrations of silica. LDH release increased in a linear, dose-dependent fashion in the range of silica doses used. The kinetics of eicosanoid release was investigated over a 3-h interval and LDH release was assayed for each time point. Within 15 min following silica addition, a shift to the production of 5-lipoxygenase metabolites was observed, accompanied by a reduction in cyclooxygenase products. This rapid alteration in AA metabolism preceded cell injury as measured by LDH release. These results demonstrate that silica is a powerful stimulator of arachidonic acid metabolism in BAM. Moreover, silica selectively stimulates the 5-lipoxygenase pathway as the dose of silica increases. Our results suggest that dysfunction in arachidonate metabolism could contribute to the pathogenesis of silicosis.

Down regulation of sodium channels in nerve terminals of spontaneously epileptic mice.

Summary1.Tottering mice, in which a single gene lesion leads to prolonged hyperexcitability and spontaneous epilepsy, were studied to determine whether enhanced electrical activity leads to down regulation of sodium channels in central neurons.2.The number of sodium channels in synaptosomes, as assessed by saxitoxin binding, was decreased from 5.38 ± 0.06 pmol/mg protein in coisogenic controls to 3.85 ± 0.10 pmol/mg protein (P < 0.001) in tottering mice without a change in theKD for saxitoxin.3.Neurotoxin-activated22Na+ influx per sodium channel was increased 80% in tottering mice (P < 0.001).4.Evidently, the increased level of electrical excitability characteristic of the tottering phenotype causes down regulation of the sodium-channel number and alteration of channel function in the nerve terminals of central neurons.

The Effects of Different Silicas on Arachidonic Acid Metabolism in Alveolar Macrophages

Bovine alveolar macrophages (BAM) prelabeled with 3H-arachidonic acid (AA) were exposed in vitro to different doses of DQ-12, Minusil-5, and Sigma silicas, or carbonyl iron beads. Arachidonic acid metabolites released into the culture medium by BAM were identified and quantitated using high performance liquid chromatography (HPLC). Cytotoxicity was assessed by the release of lactate dehydrogenase (LDH). At doses of 0.1 or 0.25 mg of DQ-12 silica and of 0.25 or 0.5 mg of Minusil-5 and Sigma silica, the release of cyclooxygenase metabolites (TXB2, PGE2, PGF2, and HHT) comprised greater than 95% of the total released AA metabolites. Silica doses above 0.5 mg led to 5-lipoxygenase metabolite release (LTB4, its two nonenzymatic isomers, and 5-HETE). This shift to 5-lipoxygenase metabolite release paralleled increased cellular cytotoxicity and was observed for each of the silicas. In contrast to silica stimulation, carbonyl iron beads elicited only small quantities of cyclooxygenase metabolites, no 5-lipoxygenase metabolites, and showed little cytotoxicity toward BAM. The relative potency of each particulate for stimulating the release of AA metabolites and LDH was calculated with DQ-12 greater than Minusil-5 greater than Sigma much greater than carbonyl iron beads. Our results indicate that the cytotoxic and presumed fibrogenic potential of a silica may be correlated with the potency to stimulate the release of 5-lipoxygenase metabolites from AM.

Arachidonic and eicosapentaenoic acid metabolism in bovine neutrophils and platelets: effect of calcium ionophore

Substitution of dietary fatty acids has potential for altering the inflammatory response. The purpose of the present study was to define the metabolites of arachidonic acid (AA) and eicosapentaenoic acid (EPA) secreted by bovine peripheral blood neutrophils and platelets. High performance liquid chromatography was used to characterize cyclooxygenase and lipoxygenase metabolites secreted in response to the calcium ionophore A23187. Cells were prelabelled with 3H‐AA or 3H‐EPA prior to challenge with the calcium ionophore. Bovine neutrophils secreted leukotriene B4 (LTB4) and 5‐hydroxyeicosatet‐raenoic acid (5‐HETE) as the major metabolites of AA, as well as the corresponding leukotriene B5 (LTB5) and 5‐hydroxyeicosapentaenoic acid (5‐HEPE) metabolites of EPA. Peptidoleukotrienes derived from 3H‐AA or 3H‐EPA were not detected under these conditions. The major tritiated metabolites secreted from bovine platelets were: thromboxane A2, measured as the stable metabolite thromboxane B2 (TXB2); hydroxyhepta‐decatrienoic acid (HHT) and 12‐HETE derived from 3H‐AA; and the omega‐3 analogs TXB3 and 12‐HEPE, derived from 3H‐EPA. Preferred substrate specificities existed amongst the AA‐ and EPA‐derived metabolites for the intermediary enzymes involved in the arachidonic acid cascade. These findings support the hypothesis that substitution of membrane‐bound AA by EPA has potential for modulation of the host inflammatory response following cellular phospholipid mobilization.

Arachidonic acid metabolism in bovine alveolar macrophages: effect of calcium ionophore on lipoxygenase products

The production of lipoxygenase metabolites of arachidonic acid was studied in bovine alveolar macrophages (BAM). Unstimulated macrophages produced small amounts of LTB4 (0.2±0.2 ng/106 BAM) but monohydroxyeicosatetraenoic acids (5t-, 12-, and 15-HETE) usually were not detectable. Both exogenous arachidonic acid and the calcium ionophore A23187 induced production of LTB4, 5-, 12-, and 15-HETE, of which 60–80% was 5-HETE. Combined challenge of BAM with both exogenous arachidonic acid and A23187 was more effective in the production of these metabolites than with either stimulus alone. The generation of the peptidoleukotrienes LTC4, LTD4, and LTE4 by BAM could not be detected under these in vitro conditions. Our results demonstrate that bovine alveolar macrophages produce similar lipoxygenase metabolites of arachidonic acid in response to A23187, as do human alveolar macrophages stimulated with the same agonist.

Seizure control following administration of anticonvulsant drugs in the quaking mouse

The effectiveness of a variety of clinical anticonvulsant drugs was evaluated in the quaking mutant mouse model of epilepsy. In this model, tonic-clonic seizures are easily elicited by handling and the effects of administration of carbamazepine (CBZ), phenytoin (DPH), phenobarbital (PB), diazepam, valproic acid (VPA) and ethosuximide were quantitatively evaluated. Chronic oral administration of CBZ, DPH and PB reduced the frequency of seizures and this was positively correlated with plasma levels of the drugs. The plasma levels of the 10,11-epoxide metabolite of CBZ were found to be approximately 3-5 times that of the parent compound with chronic oral administration. Acute intraperitoneal administration of the other drugs revealed VPA to be an effective anticonvulsant agent, whereas ethosuximide and diazepam were ineffective at dosage levels that are normally effective in mice as determined by classical testing methods such as electroshock and chemoshock. The results of the present study suggest that the quaking mouse may be a simple, reliable and inexpensive animal model for the evaluation of agents effective against focal motor seizures in humans.

Influence of extracellular calcium on the metabolism of arachidonic acid in alveolar macrophages

Bovine alveolar macrophage metabolism of arachidonic acid (AA) via cyclooxygenase (CO) and lipoxygenase (LO) pathways is stimulus specific. Under standard conditions with RPMI‐1640 media containing 0.4 mM Ca2+, the calcium ionophore A23187 stimulates the release of both CO and LO products, whereas opsonized zymosan and phorbol myristate acetate (PMA) selectively stimulate the CO pathway in these cells. We have examined the effect of varying the extracellular concentration of calcium (0–2.4 mM) on the profiles of AA metabolites secreted with differing stimuli. All stimuli caused the release of CO products, even in the absence of calcium in the media. The magnitude of release was correlated with increasing extracellular calcium concentrations, indicating some dependence of phospholipase activation on extracellular calcium. However, there were notable differences between stimuli regarding the magnitude of CO product formation and dependence on extracellular Ca2+. No 5‐LO products were demonstrable with either zymosan or PMA at any concentration of extracellular calcium tested, and inhibition of CO by indomethacin did not result in 5‐LO product formation for these stimuli. The production of 5‐LO products in bovine alveolar macrophages by A23187 required extracellular calcium, demonstrating an absolute dependence for activation of the 5‐LO pathway on an influx of extracellular calcium. Our results indicate that intracellular and extracellular Ca2+ have differing roles in the metabolism of AA down CO and LO pathways in bovine alveolar macrophages depending on the stimulus used. This regulation suggests that the pools of calcium required for activation of phospholipase A2 (PLA2) are not necessarily available for the 5‐LO enzyme.