Abstract
Neuroinflammation is a pathological hallmark of a variety of Central Nervous System (CNS) insults. Cytokines are the orchestrators of inflammatory and immune responses. Levels of tumor necrosis factor-alpha (TNF-alpha), a major proinflammatory cytokine, are upregulated following numerous CNS diseases. In addition, elevated levels of adenosine triphosphate (ATP) are observed as a result of cell damage in the CNS. In Chapter 2, utilizing an enzyme-linked immunosorbent assay (ELISA), it was confirmed that treatment of astrocytes with lipopolysaccharide (LPS), a bacterial endotoxin, induced TNF-alpha release in a concentration and time dependent manner. In Chapter 3, LPS-induced TNF-alpha release was attenuated by 1 mM ATP, a concentration known to activate P2X7 receptors. Consistent with this, 3'-O-(4-Benzoyl)benzoyl-ATP (BzATP), a P2X7 receptor agonist, also attenuated LPS-induced TNF-alpha release. This reduction in TNF-alpha release was not due to loss of cell viability. Treatment of astrocyte cultures with 10 uM or 100 uM ATP potentiated TNF-alpha release induced by a submaximal concentration of LPS. Uridine 5'-triphosphate (UTP) and 2methylthioADP (2-MeADP), P2Y receptor agonists, also enhanced this LPS-induced TNF-alpha release. In Chapter 4, pretreatment with cyclopiazonic acid (CPA), which depletes internal calcium stores, reduced LPS-induced TNF-alpha release. Also, pretreatment with 2-Aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP3) receptor inhibitor, or a high concentration of ryanodine, a caffeine/ryanodine (CAFF/RY) receptor inhibitor, reduced LPS-induced TNF-alpha release. Simultaneous pretreatment with 2-APB and ryanodine inhibited LPS-induced TNF-alpha release in an additive manner.In conclusion, Chapter 2 results suggest that LPS induces TNF-alpha release in a concentration and time dependent manner. Chapter 3 results suggest opposing effects of ATP/P2 receptor activation on TNF-alpha release, i.e., P2X receptor activation attenuates, whereas P2Y receptor activation potentiates, TNF-alpha release in LPS-stimulated astrocytes. Chapter 4 results suggest that LPS-induced TNF-alpha release involves calcium mobilization from internal stores via IP3 and CAFF/RY receptor activation and calcium influx from the extracellular space via store-operated channels and also involves the activation of ERK and p38 pathways. (Abstract shortened by UMI.)