Hyperammonemia is a key factor in the pathogenesis of hepatic encephalopathy (HE) as well as other metabolic encephalopathies, such as those associated with inherited disorders of urea cycle enzymes and in Reye's syndrome. Acute HE results in increased brain ammonia (up to 5 mM), astrocytic swelling, and altered glutamatergic function. In the present study, using fluorescence imaging techniques, acute exposure (10 min) of ammonia (NH 4 ؉ /NH 3 ) to cultured astrocytes resulted in a concentration-dependent, transient increase in [Ca 2؉ ] i . This calcium transient was due to release from intracellular calcium stores, since the response was thapsigargin-sensitive and was still observed in calcium-free buffer. Using an enzyme-linked fluorescence assay, glutamate release was measured indirectly via the production of NADH (a naturally fluorescent product when excited with UV light). Hyperammonemia consequently leads to increased concentrations of ammonia, up to 5 mM, in the brain. This high level of brain ammonia is a key factor in the pathogenesis of central nervous system dysfunction in acute and chronic liver failure. The nature and severity of the central nervous system disorder mainly depend upon the degree and acuteness of the onset of hyperammonemia (1). Acute liver failure (ALF) 1 resulting from viral infections or toxic liver injury is a life-threatening condition where hepatic encephalopathy (HE) develops rapidly and mortality rates are high due to brain stem herniation caused by increased intracranial pressure, a fatal consequence of cytotoxic brain edema. Excess ammonia is toxic to the brain resulting in deleterious effects, by both direct and indirect mechanisms, on cerebral metabolism and neurotransmission.Over the past 10 years, there has been an increasing body of evidence demonstrating that ammonia toxicity is involved in alterations of glutamatergic synaptic regulation which is implicated in the pathophysiology of HE in ALF. Several reports have consistently described increased extracellular concentrations of brain glutamate in different models of experimental ALF (2-5); however, neither the cell type nor the underlying release mechanisms have been identified. One possible explanation for the increased extracellular glutamate may be ammonia's inhibitory effects on the glutamate transporter system in astrocytes. It has been shown that ammonia inhibits glutamate uptake into astrocytes in vitro (6) and decreases protein and gene expression of the glutamate transporter GLT-1 (EAAT-2) in the frontal cortex of rats with ALF (7). The role of ammonia in the glutamatergic dysfunction demonstrated in HE is supported with a positive correlation between extracellular brain concentrations of glutamate and arterial ammonia concentrations in ALF in rats (4). In addition, using mild hypothermia as a treatment in rats with ALF, extracellular brain glutamate concentrations were normalized concomitantly with a lowering of brain ammonia (8).Glutamate has been demonstrated to be an important signaling molecule for neuro...

Zinc ions are emerging as an important factor in the etiology of neurodegenerative disorders and in brain damage resulting from ischemia or seizure activity. High intracellular levels of zinc are toxic not only to neurons but also to astrocytes, the major population of glial cells in the brain. In the present study, the role of ZnT-1 in reducing zinc-dependent cell damage in astrocytes was assessed. Zinc-dependent cell damage was apparent within 2 h of exposure to zinc, and occurred within a narrow range of approximately 200 microM. Pretreatment with sublethal concentrations of zinc rendered astrocytes less sensitive to toxic zinc levels, indicating that preconditioning protects astrocytes from zinc toxicity. Fluorescence cell imaging revealed a steep reduction in intracellular zinc accumulation for the zinc-pretreated cells mediated by L-type calcium channels. Heterologous expression of ZnT-1 had similar effects; intracellular zinc accumulation was slowed down and the sensitivity of astrocytes to toxic zinc levels was reduced, indicating that this is specifically mediated by ZnT-1 expression. Immunohistochemical analysis demonstrated endogenous ZnT-1 expression in cultured astroglia, microglia, and oligodendrocytes. Pretreatment with zinc induced a 4-fold increase in the expression of the putative zinc transporter ZnT-1 in astroglia as shown by immunoblot analysis. The elevated ZnT-1 expression following zinc priming or after heterologous expression of ZnT-1 may explain the reduced zinc accumulation and the subsequent reduction in sensitivity toward toxic zinc levels. Induction of ZnT-1 may play a protective role when mild episodes of stroke or seizures are followed by a massive brain insult.

The development of new photocleavable adenosine nucleotides based on the photochemistry of [7-(dimethylamino)coumarin-4-yl]methyl (DMACM) esters is described. The phototriggers liberate adenosine triphosphate (ATP), diphosphate, and monophosphate upon UV/Vis irradiation between 334 and 405 nm. The efficiency of photocleavage at long wavelengths is high as a result of a combination of appropriate quantum yields and intensive absorptivities. By using time-resolved fluorescence spectroscopy, we determined a lower limit of 1.6 x 10(9) s(-1) for the rate constant of the release of ATP from DMACM-caged ATP. The favorable properties of DMACM-caged ATP were confirmed in physiological studies by confocal laser scanning microscopy. We were able to uncage DMACM-caged ATP in cultures of mouse astrocytes and in brain tissue slices from mice and were also able to measure the effect of photoreleased ATP on the cellular response of astrocytes, namely the ability of the ATP to evoke Ca(2+) ion waves.

Astrocytes express a variety of metabotropic receptors and their activation leads to a biphasic Ca2+ response due to Ca2+ release from intracellular stores and subsequent capacitative Ca2+ entry. We performed Ca2+ imaging with Fura-2 on cultured mouse astrocytes and showed that extracellular zinc reversibly blocks the capacitative Ca2+ entry following application of the metabotropic ligands ATP, glutamate and endothelin-1. Zinc blocked the plateau phase of the ligand-triggered Ca2+ responses. When ligands were repetitively applied in the presence of zinc the calcium responses progressively decayed and even disappeared, indicating that capacitative Ca2+ entry is required to refill the stores. Zinc inhibited the capacitative Ca2+ entry with a K(i) of approximately 6 microM, which is well within the physiological concentration range of zinc found in the brain. Application of the reducing agent DTT prevented the blocking effect by zinc ions but not the inhibition elicited by the nonphysiological metal ions Gd3+ and La3+, indicating that zinc has a distinct binding site. To monitor the capacitative Ca2+ entry in astrocytes in situ and to determine the effect of zinc on this pathway we utilized X-rhod-1 imaging in hippocampal slices of a transgenic mouse line with green fluorescent astrocytes. Zinc affected the repetitive metabotropic Ca2+ response in the following fashion: (i) after depleting stores in Ca(2+)-free solution, re-addition of Ca2+ led to an influx of Ca2+ via a zinc-sensitive Ca2+ entry route; (ii) with repetitive application of metabotropic ligands, Ca2+ responses became smaller and even disappeared in the presence of zinc. We conclude that zinc, which is co-released from glutamatergic synaptic vesicles upon neuronal activity, has a major impact on shaping the astrocytic calcium responses.

Hypoxic damage to the central nervous system triggers morphological and functional responses in astrocytes (AC). When isolated from the cerebral microenvironment and placed in cell culture, hypoxia promotes astrocytic alterations indicative of dedifferentiation. To investigate the effect of hypoxia on AC morphology, we performed studies on cultured AC pretreated with dibutyrylcAMP. These treated cells resemble AC in vivo, assuming a stellate morphology as observed by phase contrast microscopy. Exposure to hypoxia (0.3% oxygen tension) for 24 h induced a flat and polygonal shape in most of the cells as opposed to normoxic controls. A candidate factor to mediate this response is endothelin-1 (ET-1), a peptide produced by ischemic AC in vivo. The role of the astrocytic ET system during hypoxia, therefore, was investigated. Exogenous application of ET-1 mimicked the effect of hypoxia on the astrocytic morphology. The effects of hypoxia and exogenous ET-1 on the morphology were inhibited by the nonselective ET receptor antagonists PD142893 and PD145065. The ET peptide levels in the culture supernatants of AC increased about 1.5-fold after 24 h of hypoxia as measured by radioimmunoassay. Northern blot analyses revealed a threefold up-regulation of prepro ET-1 mRNA and a concomitant downregulation of ET A receptor and ET B receptor mRNAs. However, calcium responses were still inducible by exogenous ET-1. These results indicate that hypoxia triggers an autocrine loop in AC, resulting in a morphological transformation. This response is independent from neuronal damage and is based on activation of the astrocytic ET system. Key words: cell morphology • astrocytes • cell morphology • cultured cells • culture media • endothelin-1 • endothelin converting enzyme ypoxia is a very common pathophysiological condition of the brain, which is either isolated, for example, in neonatal asphyxia, or in combination with substrate deprivation, as in ischemia. Many authors have discussed the morphology of astrocytes (AC) in a brain that has been subjected to ischemia or hypoxia. Loss of astrocytic processes during global cerebral ischemia was first recognized by Alzheimer in 1910 (1). He noted that AC could undergo a process he termed "clasmatodendrosis", which results in these cells assuming an H amoeboid shape with a clear reduction in surface area. Clasmatodendrosis can be observed as early as 30 min following occlusion of the middle cerebral artery in the rat (2). On the ultrastructural level, astrocytic processes disappear from between adjacent dendrites with the possible consequence of increased neurotransmitter levels at synapses of compromised neurons (3,4). During ischemia these alterations might favor excitotoxic neuronal death.Considering the clinical relevance of factors influencing the outcome of ischemia, it is of great importance to elucidate the mechanisms evoking these early astrocytic responses, as pharmacological intervention might be capable of prolonging postischemic neuronal survival. Cell culture models provide a va...