Title

Characterization of TNF-alpha release in LPS-stimulated astrocytes: Bi-functional effects of purinergic receptor activation and role for calcium signaling

Date of Award

2005

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Neuroscience

First Committee Member

Joseph T. Neary, Committee Chair

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.)

Keywords

Biology, Neuroscience

Link to Full Text

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