Actinobacillus actinomycetemcomitans leukotoxin (LTX) is a cytotoxic protein which kills human leukocytes. Upon the addition of low concentrations of LTX, target cells undergo an immediate and sustained increase in intracellular calcium, followed by downstream apoptotic events such as perturbation in mitochondrial function, caspase activation, DNA fragmentation and cell death. Objective: The purpose of this study is to examine the role of the inositol 1,4,5-triphosphate receptor (IP₃R1) in LTX mediated cytotoxicity. One source of elevated calcium is the endoplasmic reticulum intracellular calcium store. Our preliminary studies have indicated that blocking Ca²⁺ release from the endoplasmic reticulum (ER) by preincubation with ryanodine prevents [Ca²⁺]_c increases and suggests involvement of calcium channels, such as the IP₃ receptor or ryanodine receptor, in the ER. In addition using reverse transcriptase PCR, we found that our transfectant that exhibits upregulated BclX_L expression and inhibits LTX-mediated cytotoxicity and apoptosis also has decreased IP₃R1 message. We therefore asked if deletion of IP₃R1 blocks LTX mediated cytotoxicity. Methods: Jurkat cells, transfected with an IP₃R1 antisense fragment, were exposed to 10^-9 M LTX and cytotoxicity measured via trypan blue exclusion. Results: LTX killed 45.54% of control cells compared to 4.88% of cells exhibiting unregulated antisense IP₃R1 expression and 49.34% for Jurkats. The kill was inhibitable with heat and antibody. Conclusion: IP₃R1 appears to play a role in LTX mediated cytotoxicity.

Supported by DE12305.

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