Hypoxia reverses dibutyryl‐cAMP‐induced stellation of cultured astrocytes via activation of the endothelin system

Abstract

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...

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