CORESTA Meeting, Smoke Science/Product Technology, 2017, Kitzbühel, STPOST 19

Combustible and smokeless tobacco product preparations differentially regulate calcium mobilization in the human leukemic HL60 cell line

MAKENA P.(2); ARIMILLI S.(1); DAMRATOSKI B.E.(1); PRASAD G.L.(2)
(1) Wake Forest University School of Medicine, Department of Microbiology & Immunology, Winston-Salem, NC, U.S.A.; (2) RAI Services Co., Winston-Salem, NC 27102, U.S.A.

Increased calcium (Ca2+) mobilization is central to leukocyte signaling and for regulation of immune responses. Although evidence suggests that cigarette smoking affects several biological functions including inflammatory responses, how the use of combustible (cigarette) and smokeless tobacco regulates Ca2+ mobilization and consequently inflammation is unclear. We evaluated the effects of several Tobacco Product Preparations (TPPs) including total particulate matter (TPM) from 3R4F reference cigarettes, phosphate buffer extracts of 2S3 moist snuff (as a representative of smokeless tobacco [ST]) and nicotine on Ca2+ mobilization in Human Leukemic HL60 cells. HL60 cells were initially loaded with Flow-3, a Ca2+ indicator dye and then treated with different equi-nicotine units of TPM (1, 5, 10, and 20 µg/mL) or ST (100, 150, 200 and 250 µg/mL), or nicotine (0, 10, 50, and 100 µg/mL). Intracellular Ca2+ mobilization (Ca2+)I was measured using flow cytometry. TPM treatment significantly increased (Ca2+)I in a concentration dependent manner, but not treatment with ST or nicotine. The induction of TPM-mediated (Ca2+)I occurred at lower nicotine equivalent units compared to ST or nicotine. Experiments using Thapsigargin (TG) for depletion of Endoplasmic Reticulum (ER) Ca2+ stores suggested that TPM-mediated (Ca2+)I induction occurs through multiple Ca2+ stores including ER stores. Assessment of extracellular Ca2+ mobilization (Ca2+)E using CaCl2 in intact or TG treated cells for depletion of ER Ca2+ stores indicated that TPM-can mobilize CaCl2 as an extracellular source of Ca2+ and induce (Ca2+)E. These results, for the first time, demonstrate that combustible TPPs, such as TPM, trigger both intracellular Ca2+ release and also elicit (Ca2+)E by capacitative (in depleted ER stores) and non-capacitative (in intact ER stores) Ca2+ mobilization. On the other hand, Ca2+ mobilization was unaffected by ST or nicotine treatment under the experimental conditions. In conclusion, our results suggest that TPM drives intra- and extracellular Ca2+ mobilization that may activate signaling pathways potentially leading to altered inflammatory responses in leukocytes.