The role of PIP2 and the IP3/DAG pathway in intracellular calcium release and cell survival during nanosecond electric pulse exposures

Zachary A. Steelman; Gleb P. Tolstykh; Larry E. Estlack; Caleb C. Roth; Bennett L. Ibey

doi:10.1117/12.2079928

12 March 2015 The role of PIP₂ and the IP₃/DAG pathway in intracellular calcium release and cell survival during nanosecond electric pulse exposures

Zachary A. Steelman, Gleb P. Tolstykh, Larry E. Estlack, Caleb C. Roth, Bennett L. Ibey

Author Affiliations +

Proceedings Volume 9326, Energy-based Treatment of Tissue and Assessment VIII; 932611 (2015) https://doi.org/10.1117/12.2079928
Event: SPIE BiOS, 2015, San Francisco, California, United States

Abstract

Phosphatidylinositol_4,5-biphosphate (PIP₂) is a membrane phospholipid of particular importance in cell-signaling pathways. Hydrolysis of PIP₂ releases inositol-1,4,5-triphosphate (IP₃) from the membrane, activating IP₃ receptors on the smooth endoplasmic reticulum (ER) and facilitating a release of intracellular calcium stores and activation of protein kinase C (PKC). Recent studies suggest that nanosecond pulsed electric fields (nsPEF) cause depletion of PIP₂ in the cellular membrane, activating the IP₃ signaling pathway. However, the exact mechanism(s) causing this observed depletion of PIP₂ are unknown. Complicating the matter, nsPEF create nanopores in the plasma membrane, allowing calcium to enter the cell and thus causing an increase in intracellular calcium. While elevated intracellular calcium can cause activation of phospholipase C (PLC) (a known catalyst of PIP₂ hydrolysis), PIP₂ depletion has been shown to occur in the absence of both extracellular and intracellular calcium. These observations have led to the hypothesis that the high electric field itself may be playing a direct role in the hydrolysis of PIP2 from the plasma membrane. To support this hypothesis, we used edelfosine to block PLC and prevent activation of the IP₃/DAG pathway in Chinese Hamster Ovarian (CHO) cells prior to applying nsPEF. Fluorescence microscopy was used to monitor intracellular calcium bursts during nsPEF, while MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survivability assays were utilized to determine whether edelfosine improved cell survival during nsPEF exposure. This work is critical to refine the role of PIP₂ in the cellular response to nsPEF, and also to determine the fundamental biological effects of high electric field exposures.

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Zachary A. Steelman, Gleb P. Tolstykh, Larry E. Estlack, Caleb C. Roth, and Bennett L. Ibey "The role of PIP₂ and the IP₃/DAG pathway in intracellular calcium release and cell survival during nanosecond electric pulse exposures", Proc. SPIE 9326, Energy-based Treatment of Tissue and Assessment VIII, 932611 (12 March 2015); https://doi.org/10.1117/12.2079928

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