Combustible Cigarette and Smokeless Tobacco Product Preparations Differentially Regulate Intracellular Calcium Mobilization in HL60 Cells

Changes in the level of intracellular calcium ([Ca²⁺]_i) are central to leukocyte signaling and immune response. Although evidence suggests that cigarette smoking affects inflammatory response via an increase in intracellular calcium, it remains unclear if the use of smokeless tobacco (e.g., moist snuff) elicits a similar response. In this study, we evaluated the effects of tobacco product preparations (TPPs), including total particulate matter (TPM) from 3R4F reference cigarettes, smokeless tobacco extract (STE) from 2S3 reference moist snuff, and nicotine alone on Ca²⁺ mobilization in HL60 cells. Treatment with TPM, but not STE or nicotine alone, significantly increased [Ca²⁺]_i in a concentration-dependent manner in HL60 cells. Moreover, TPM-induced [Ca²⁺]_i increase was not related to extracellular Ca²⁺ and did not require the activation of the IP3 pathway nor involved the transient receptor potential (TRP) channels. Our findings indicate that, in cells having either intact or depleted endoplasmic reticulum (ER) Ca²⁺ stores, TPM-mediated [Ca²⁺]_i increase involves cytosolic Ca²⁺ pools other than thapsigargin-sensitive ER Ca²⁺ stores. These results, for the first time, demonstrate that TPM triggers [Ca²⁺]_i increases, while significantly higher nicotine equivalent doses of STE or nicotine alone, did not affect [Ca²⁺]_i under the experimental conditions. In summary, our study suggests that in contrast with STE or nicotine preparations, TPM activates Ca²⁺ signaling pathways in HL60 cells. The differential effect of combustible and non-combustible TPPs on Ca²⁺ mobilization could be a useful in vitro endpoint for tobacco product evaluation.