Sean Murphy

Induction of Nitric Oxide Synthase in Glial Cells

Abstract: Primary astrocyte cultures, C6 glioma cells, and N18 neuroblastoma cells were assayed for nitric oxide synthase (NOS) activity with a bioassay of cyclic GMP production in RFL‐6 fibroblasts. Treatment of astrocyte cultures for 16–18 h with lipopolysaccharide (LPS) induced NOS‐like activity that was l‐arginine and NADPH dependent, Ca2+ independent, and potentiated by superoxide dismutase. Induction was evident after 4 h, was dependent on the dose of LPS, and required protein synthesis. Treatment of astrocyte cultures with leucine methyl ester reduced microglial cell contamination from 7 to 1%, with a loss of 44% of NOS‐like activity. C6 cells treated with LPS also showed Ca2+‐independent and l‐arginine‐dependent NOS‐like activity. N18 cells demonstrated constitutive Ca2+‐dependent NOS‐like activity that was not enhanced by LPS induction. These data indicate that NOS‐like activity can be induced in microglia, astrocytes, and a related glioma cell line as it can in numerous other cell types, but not in neuron‐like N18 cells.

The possible role of glia in nociceptive processing and hyperalgesia in the spinal cord of the rat

Recent studies have suggested that glia might play a more active role in synaptic function than previously thought. Therefore, the present studies have evaluated the potential role of spinal cord glia in acute nociceptive processing and in the thermal and mechanical hyperalgesia produced by peripheral injury. In the present experiments, we found that: (1) selective inhibition of glia metabolism with intrathecal (i.t.) administration of fluorocitrate (1 nmol) results in a marked, but reversible, attenuation of the persistent thermal and mechanical hyperalgesia produced by intraplantar zymosan (5 mg); (2) selective inhibition of the inducible form of nitric oxide synthase (iNOS) with i.t. aminoguanidine (1 pmol-1 nmol) resulted in a dose-dependent inhibition of the persistent thermal, but not mechanical hyperalgesia produced by intraplantar zymosan (5 mg); (3) i.t. coadministration of interleukin 1 beta (IL1 beta; 10 ng) and interferon gamma (IFN; 1000 U) resulted in expression of the message for iNOS 8 hr after administration assessed using reverse-transcription polymerase chain reaction (RT-PCR) and Southern blot analysis; and (4) i.t. administration of lipopolysaccharide (LPS; 150 micrograms) produced a time-dependent thermal hyperalgesia compared with saline treated-rats (15 microliters). There was no change in mechanical withdrawal thresholds over time following any treatment, except fluorocitrate. We have previously shown that NO plays a significant role in mechanisms of hyperalgesia. In the present experiments we have extended these observations and have now shown a role for iNOS, expressed by glia, in mechanisms of hyperalgesia. These results suggest an unexplored avenue for the development of potential new and novel therapies for pain control.

Production of nitric oxide by glial cells: Regulation and potential roles in the CNS

Roles proposed for nitric oxide (NO) in CNS pathophysiology are increasingly diverse and range from intercellular signaling, through necrotic killing of cells and invading pathogens, to the involvement of NO in apoptosis and tissue remodeling. In vitro evidence and observations from experimental animal models of a variety of human neuropathologies, including stroke, indicate that glial cells can produce NO. Regulation of at least one of the NO synthase genes (NOS‐2) in glia has been well described; however, apart from hints emerging out of co‐culture studies and extrapolation based upon the reactivity of NO, we are a long way from identifying functions for glial‐derived NO in the CNS. Although the assumption is that NO is very often cytotoxic, it is evident that NO production does not always equate with tissue damage, and that both the cellular source of NO and the timing of NO production are important factors in terms of its effects. With the development of strategies to transfer or manipulate expression of the NOS genes in specific cells in situ, the ability to deliver NO into the CNS via long‐lived chemical donors, and the emergence of more selective NOS inhibitors, an appreciation of the significance of glial‐derived NO will change. GLIA 29:1–13, 2000.

Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:6ω-3) and Arachidonic Acid (20:4ω-6)

Abstract: Elongated, highly polyunsaturated derivatives of linoleic acid (18:2ω‐6) and linolenic acid (18:3ω‐3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1‐14C]18:2ω‐6, [1‐14C]20:4ω‐6, [1‐14C]18:3ω‐3, or [1‐14C]20:5ω‐3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of Δ4‐desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18‐ and 20‐carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18‐ and 20‐carbon precursors. The major metabolite of 18:2ω‐6 was 20:4ω‐6, whereas the primary products from 18:3ω‐3 were 20:5ω‐3, 22:5ω‐3, and 22:6ω‐3. The majority of the long‐chain fatty acids formed by astrocyte cultures, particularly 20:4ω‐6 and 22:6ω‐3, was released into the extracellular fluid. Although incapable of producing 20:4ω‐6 and 22:6ω‐3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating ω‐6 and ω‐3 essential fatty acid precursors to 20:4ω‐6 and 22:6ω‐3, then releasing the long‐chain polyunsaturated fatty acids for uptake by neurons.

Evidence for an Astrocyte‐Derived Vasorelaxing Factor with Properties Similar to Nitric Oxide

Abstract: To determine whether astrocytes release nonprostanoid vasodilators, cells on microcarrier beads were superfused with various agents in the presence of indomethacin, and the effluent was bioassayed and also analyzed for nitric oxide by a chemiluminescence technique. Bradykinin and A23187 induced release of a factor that relaxed arterial rings, an effect that was blocked by hemoglobin. The effluent contained either nitric oxide or a related compound that could be reduced to nitric oxide. Production of this factor was competitively inhibited by the arginine analogs NG‐nitro‐L‐arginine and NG‐methyl‐L‐arginine and could be restored with L‐arginine. Quisqualate and norepinephrine were also effective in causing the release of nitric oxide from astroglial cells. Thus, astrocyte‐derived relaxing factor has properties similar to those of an endothelium‐ and neuronderived relaxing factor.

Astrocyte glutamate receptor activation promotes inositol phospholipid turnover and calcium flux

Astrocyte-enriched cultures prepared from the neonatal rat cortex were prelabelled with either [3H]myoinositol or 45Ca2+ and then exposed to various excitatory amino acids. This resulted in an increase in both the breakdown of membrane inositol phospholipids and Ca2+ flux with the following rank order of efficacy: quisqualate greater than or equal to glutamate (Glu) greater than kainate much greater than N-methyl-D-aspartate. Experiments performed with the Ca2+ ionophore A23187 and in the absence of medium Ca2+ suggested that Glu-evoked 45Ca2+ efflux was primarily the result of an increased influx of extracellular Ca2+. However, Glu-stimulated inositol lipid metabolism was found to be only partially dependent on extracellular Ca2+. The quisqualate-preferring receptor antagonist gamma-glutamylaminomethylsulphonic acid was found to be effective in reversing both Glu-evoked inositol lipid breakdown and Ca2+ flux. The results presented are suggestive of some form of interaction between Glu receptors coupled to inositol lipid turnover and Ca2+ channel opening in astrocytes.

ATP‐Evoked Ca2+ Mobilisation and Prostanoid Release from Astrocytes: P2‐Purinergic Receptors Linked to Phosphoinositide Hydrolysis

Abstract: Astrocyte cultures prelabelled with either [3H]‐inositol or 45Ca2+ were exposed to ATP and its hydrolysis products. ATP and ADP, but not AMP and adenosine, produced increases in the accumulation of intracellular 3H‐labelled inositol phosphates (IP), efflux of 45Ca2+, and release of thromboxane A2 (TXA2). Whereas ATP‐stimulated 3H‐IP accumulation was unaffected, its ability to promote TXA2 release was markedly reduced by mepacrine, an inhibitor of phospholipase A2 (PLA2). ATP‐evoked 3H‐IP production was also spared following treatment with the cyclooxygenase inhibitor, indomethacin. We conclude that ATP‐induced phosphoinositide (PPI) breakdown and 45Ca2+ mobilisation occurred in parallel with, if not preceded, the release of TXA2. Following depletion of intracellular Ca2+ with a brief preexposure to ATP in the absence of extracellular Ca2+, the release of TXA2 in response to a subsequent ATP challenge was greatly reduced when compared with control. These results suggest that mobilisation of cytosolic Ca2+ may be the stimulus for PLA2 activation and, thus, TXA2 release. Stimulation of α‐adrenoceptors also caused PPI breakdown and 45Ca2+ efflux but not TXA2 release. The effects of ATP and noradrenaline (NA) on 3H‐IP accumulation were additive, but their combined ability to increase 45Ca2+ efflux was not. Interestingly, in the presence of NA, ATP‐stimulated TXA2 release was reduced. Our data provide evidence that functional P2‐purinergic receptors are present on astrocytes and that ATP is the first physiologically relevant stimulus found to initiate prostanoid release from these cells.

G-CSF reduces infarct volume and improves functional outcome after transient focal cerebral ischemia in mice

Growth factors possess neuroprotective and neurotrophic properties in vitro, but few have been extensively studied in vivo after stroke. In the present study, we investigated the potential functional benefits of granulocyte colony-stimulating factor (G-CSF) administration after focal cerebral ischemia. Male mice underwent 60-minute middle cerebral artery occlusion (MCAO) and received G-CSF (50 μg/kg, subcutaneously) or vehicle (saline) at the onset of reperfusion. Granulocyte colony-stimulating factor-treated mice killed at 48 hours after MCAO revealed a >45% reduction (P<0.05) in lesion volume. In terms of body weight recovery, and in tests of motor (grid test and rotarod) and cognitive ability (water maze), MCAO significantly worsened the outcome in vehicle-treated mice as compared with shams (P<0.05). However, G-CSF treatment was beneficial as, compared with vehicle, this significantly improved weight recovery and motor ability. This effect was most apparent on the water maze where G-CSF-treated mice were indistinguishable from shams in terms of acquiring the task. These results indicate long-term beneficial effects of a single dose of G-CSF administered on reperfusion, and illustrate the need to further investigate the mechanisms of G-CSF action.

Activation of Muscarinic and of α1‐Adrenergic Receptors on Astrocytes Results in the Accumulation of Inositol Phosphates

Abstract: Astrocyte‐enriched cultures prepared from the newborn rat cortex incorporated [3H]myo‐inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose‐dependent and blocked by specific α1‐adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+ ‐free medium, but was abolished when EGTA was also present in the Ca2+ ‐free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo‐inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.

Progesterone suppresses the inflammatory response and nitric oxide synthase-2 expression following cerebral ischemia.

Gender differences in outcome following cerebral ischemia have frequently been observed and attributed to the actions of steroid hormones. Progesterone has been shown to possess neuroprotective properties following transient ischemia, with respect to decreasing lesion volume and improving functional recovery. The present study was designed to determine the mechanisms of progesterone neuroprotection, and whether these relate to the inflammatory response. Male mice underwent either 60 min or permanent middle cerebral artery occlusion (MCAO) and received progesterone (8 mg/kg ip) or vehicle 1 h, 6 h and 24 h post-MCAO. Forty-eight hours following transient MCAO, structural magnetic resonance imaging revealed a significant decrease in the amount of edematous tissue present in progesterone-treated mice as compared with vehicle. Using real-time PCR we found that progesterone treatment significantly suppressed the injury-induced upregulation of interleukin (IL)-1beta, transforming growth factor (TGF)beta2, and nitric oxide synthase (NOS)-2 mRNAs in the ipsilateral hemisphere while having no effect on tumor necrosis factor (TNF)-alpha mRNA expression. Progesterone treatment following permanent MCAO also resulted in a significant decrease in lesion volume. This was not apparent in mice lacking a functional NOS-2 gene. Thus, progesterone is neuroprotective in both permanent and transient ischemia, and this effect is related to the suppression of specific aspects of the inflammatory response.