Thrombospondin (TSP)-1, a multidomain glycoprotein, is secreted from astrocytes and promotes synaptogenesis. However, little is known about the mechanisms regulating its expression and release. In this article, we report that purinergic signaling participates in the production and secretion of TSP-1. Treatment of primary cultures of rat cortical astrocytes with extracellular ATP caused an increase in TSP-1 expression in a time-and concentration-dependent manner and was inhibited by antagonists of P2 and P1 purinergic receptors. Agonist studies revealed that UTP, but not 2,3-O-(4-benzoyl)benzoyl-ATP, 2-methylthio-ADP, adenosine, or 5-N-ethyl-carboxamidoadenosine, caused a significant increase in TSP-1 expression. In addition, release of TSP-1 was stimulated by ATP and UTP but not by 2-methylthio-ADP or adenosine. Additional studies indicated that P2Y 4 receptors stimulate both TSP-1 expression and release. P2Y receptors are coupled to protein kinase cascades, and signaling studies demonstrated that blockade of mitogen-activated protein kinases or Akt inhibited ATP-and UTPinduced TSP-1 expression. Using an in vitro model of CNS trauma that stimulates release of ATP, we found that TSP-1 expression increased after mechanical strain and was completely blocked by a P2 receptor antagonist and by inhibition of p38͞mitogen-activated protein kinase and Akt, thereby indicating a major role for P2 receptor͞protein kinase signaling in TSP-1 expression induced by trauma. We conclude that TSP-1 expression can be regulated by activation of P2Y receptors, particularly P2Y 4, coupled to protein kinase signaling pathways and suggest that purinergic signaling may be an important factor in TSP-1-mediated cell-matrix and cell-cell interactions such as those occurring during development and repair.neuron-glia interaction ͉ traumatic injury ͉ nucleotide receptors ͉ gliosis ͉ protein kinase R ecent evidence has shifted the focus on astrocytes from primarily a passive role involved in homeostasis to a more active role in a number of key physiological and pathological interactions with neurons (1-4). One of the ways in which astrocytes can interact with neurons is by the expression or release of recognition molecules. Thrombospondins (TSPs) are large multimeric, multidomain glycoproteins that participate in cell-cell and cell-matrix interactions (5). TSPs are expressed in many tissues, but their importance in the CNS is just beginning to be understood. For instance, two recent studies have focused attention on the role of TSPs in CNS development and repair. Christopherson et al. (6) showed that application of purified TSP-1, TSP-2, or astrocyte-conditioned medium was sufficient to increase the number of synapses in retinal ganglion cells and that these synapses were presynaptically active. In addition, Lin et al. (7) reported that after ischemia, increased levels of TSP-1 were localized in astrocytes and endothelial cells near blood vessels. These studies indicate the importance of TSPs in synapse formation during development and in remo...

1 Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X 7 subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. 2 P2Y and P2X receptor agonists ATP, uridine 5 0 -triphosphate (UTP) and 2 0 ,3 0 -O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration-and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X 7 receptor. Activation was maximal at 5-10 min and was sustained for 60 min; the EC 50 for BzATP was approximately 50 mM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. 3 The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X 7 receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5 0 -phosphate-6-azophenyl-2 0 ,4 0 -disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. 4 Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. 5 In conclusion, our data indicate that stimulation of astrocytic P2X 7 receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism.

Protein kinase B/Akt is a key signaling molecule that regulates cell survival, growth, and metabolism, and inhibits apoptosis. Traumatic brain injury (TBI) activates Akt, and Akt has been implicated in neuronal survival after TBI, but little is known about injury-induced Akt activation in astrocytes, cells that exhibit hypertrophic and hyperplastic responses to CNS injury. Here we have investigated the effect of mechanical strain on Akt activation in primary cultures of rat cortical astrocytes growing on deformable Silastic membranes. When astrocytes were subjected to mechanical strain (50 msec; 5-7.5 mm displacement), we observed an increase in phosphorylation of serine 473, a key indicator of Akt activation. Akt phosphorylation was increased at 3 min postinjury, was maximal from 5 to 10 min, and declined gradually thereafter. Akt activation was also dependent on the severity of the injury. Stretch-induced Akt phosphorylation was attenuated by blocking calcium influx and phosphoinositide 3-kinase (PI3K), an upstream activator of Akt. In addition, we found that ATP is rapidly released after mechanical strain and that the P2 purinergic receptor antagonist iso-pyridoxal-5'-phosphate-6-azophenyl-2',5'disulfonate (PPADS) attenuated trauma-induced Akt activation. We conclude that mechanical strain causes activation of Akt in astrocytes via stimulation of P2 receptors. This suggests that P2 receptor/Akt signaling promotes astrocyte survival and growth, and this process may play a role in the generation of reactive gliosis after TBI.

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Aquaporin-4 water channels play a central role in brain water regulation in neurological disorders. Aquaporin-4 is abundantly expressed at the astroglial endfeet facing the cerebral vasculature and the pial membrane, and both its expression level and subcellular localization significantly influence brain water transport. However, measurements of aquaporin-4 levels in animal models of brain injury often report opposite trends of change at the injury core and the penumbra. Furthermore, aquaporin-4 channels play a beneficial role in brain water clearance in vasogenic edema, but a detrimental role in cytotoxic edema and exacerbate cell swelling. In light of current evidence, we still do not have a complete understanding of the role of aquaporin-4 in brain water transport. In this review, we propose that the regulatory mechanisms of aquaporin-4 at the transcriptional, translational, and post-translational levels jointly regulate water permeability in the short and long time scale after injury. Furthermore, in order to understand why aquaporin-4 channels play opposing roles in cytotoxic and vasogenic edema, we discuss experimental evidence on the dynamically changing osmotic gradients between blood, extracellular space, and the cytosol during the formation of cytotoxic and vasogenic edema. We conclude with an emerging picture of the distinct osmotic environments in cytotoxic and vasogenic edema, and propose that the directions of aquaporin-4-mediated water clearance in these two types of edema are distinct. The difference in water clearance pathways may provide an explanation for the conflicting observations of the roles of aquaporin-4 in edema resolution.