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Cell swelling and blebbing has been commonly observed following nanosecond pulsed electric field (nsPEF) exposure. The hypothesized origin of these effects is nanoporation of the plasma membrane (PM) followed by transmembrane diffusion of extracellular fluid and disassembly of cortical actin structures. This investigation will provide evidence that shows passive movement of fluid into the cell through nanopores and increase of intracellular osmotic pressure are not solely responsible for this observed phenomena. We demonstrate that phosphatidylinositol-4,5-bisphosphate (PIP2) depletion and hydrolysis are critical steps in the chain reaction leading to cellular blebbing and swelling. PIP2 is heavily involved in osmoregulation by modulation of ion channels and also serves as an intracellular membrane anchor to cortical actin and phospholipase C (PLC). Given the rather critical role that PIP2 depletion appears to play in the response of cells to nsPEF exposure, it remains unclear how its downstream effects and, specifically, ion channel regulation may contribute to cellular swelling, blebbing, and unknown mechanisms of the lasting “permeabilization” of the PM.

Abstract: Loss of venom from the venom gland after biting or manual extraction leads to morphological changes in venom secreting cells and the start of a cycle of production of new venom. We have previously shown that stimulation of both α- and β-adrenoceptors in the secretory cells of the venom gland is essential for the onset of the venom production cycle in Bothrops jararaca. We investigated the signaling pathway by which the α-adrenoceptor initiates the venom production cycle. Our results show that the α1-adrenoceptor subtype is present in venom gland of the snake. In quiescent cells, stimulation of α1-adrenoceptor with phenylephrine increased the total inositol phosphate concentration, and this effect was blocked by the phospholipase C inhibitor U73122. Phenylephrine mobilized Ca2+ from thapsigargin-sensitive stores and increased protein kinase C activity. In addition, α1-adrenoceptor stimulation increased the activity of ERK 1/2, partially via protein kinase C. Using RT-PCR approach we obtained a partial sequence of a snake α1-adrenoceptor (260 bp) with higher identity with α1D and α1B-adrenoceptors from different species. These results suggest that α1-adrenoceptors in the venom secreting cells are probably coupled to a Gq protein and trigger the venom production cycle by activating the phosphatidylinositol 4,5-bisphosphate and ERK signaling pathway. [Copyright &y& Elsevier]

Summary Leukemia stem cells (LSCs) are a rare subpopulation of abnormal hematopoietic stem cells (HSCs) that propagates leukemia and are responsible for the high frequency of relapse in therapies. Detailed insights into LSCs' survival will facilitate the identification of targets for therapeutic approaches. Here, we develop an inhibitor, LYZ-81, which targets ORP4L with high affinity and specificity and selectively eradicates LCSs in vitro and in vivo. ORP4L is expressed in LSCs but not in normal HSCs and is essential for LSC bioenergetics and survival. It extracts PIP 2 from the plasma membrane and presents it to PLCβ3, enabling IP 3 generation and subsequent Ca2+-dependent bioenergetics. LYZ-81 binds ORP4L competitively with PIP 2 and blocks PIP 2 hydrolysis, resulting in defective Ca2+ signaling. The results provide evidence that LSCs can be eradicated through the inhibition of ORP4L by LYZ-81, which may serve as a starting point of drug development for the elimination of LSCs to eventually cure leukemia. Graphical Abstract Highlights • ORP4L is highly expressed in LSCs and essential for LSC survival • ORP4L extracts and presents PIP 2 from the plasma membrane for PLCβ3 catalysis in LSCs • LYZ-81 is identified as a specific inhibitor of ORP4L • Targeting ORP4L by LYZ-81 selectively eradicates LSCs Zhong et al. report that abnormal expression of ORP4L is essential for leukemia stem cell survival; it enables IP 3 generation by extracting and presenting PIP 2 from the plasma membrane to PLCβ3 for hydrolysis. The compound LYZ-81, which blocks this process via targeting ORP4L, selectively eradicates leukemia stem cells. [ABSTRACT FROM AUTHOR]