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

Abstract

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.

Keywords: calcium mobilization; combustible; non-combustible; tobacco product preparations.

Fig. 1

Effect of TPPs on [Ca²⁺]_i. Fluo-3AM–labeled HL60 cells exposed to equi-nicotine units of TPM and STE or different concentrations of nicotine. Intracellular Ca²⁺ mobilization was measured using flow cytometry. PMA/ionomycin was used as a positive control and DMSO and PB as negative vehicle controls for TPM and STE, respectively. Normalized AUC of PMA/ionomycin and different concentrations of STE, TPM, and nicotine from three independent experiments are shown as mean ± SD (a). A linear regression was performed for each treatment to test the dose effect on the [Ca²⁺]_i; the statistical significance is indicated by *p < 0.05. Flow cytometer raw data of kinetics from HL60 cells exposed to different concentrations of TPM (b), STE (c), and nicotine (d).

Fig. 1

Effect of TPPs on [Ca²⁺]_i. Fluo-3AM–labeled HL60 cells exposed to equi-nicotine units of TPM and STE or different concentrations of nicotine. Intracellular Ca²⁺ mobilization was measured using flow cytometry. PMA/ionomycin was used as a positive control and DMSO and PB as negative vehicle controls for TPM and STE, respectively. Normalized AUC of PMA/ionomycin and different concentrations of STE, TPM, and nicotine from three independent experiments are shown as mean ± SD (a). A linear regression was performed for each treatment to test the dose effect on the [Ca²⁺]_i; the statistical significance is indicated by *p < 0.05. Flow cytometer raw data of kinetics from HL60 cells exposed to different concentrations of TPM (b), STE (c), and nicotine (d).

Fig. 2

Effect of TPPs on extracellular Ca²⁺ influx. Fluo-3AM–labeled HL60 cells exposed to equi-nicotine units of TPM and STE. a Single concentration of CaCl₂ was added to each concentration of TPP. Intracellular Ca²⁺ mobilization, [Ca²⁺]_i, was measured using flow cytometry. DMSO and PB were used as negative vehicle controls for TPM and STE, respectively. Normalized AUC of PMA/ionomycin and different concentrations of TPM and STE from three independent experiments are shown as mean ± SD. For each TPP, two-way analysis of variance and post hoc analysis using Tukey’s honestly significant difference test (HSD) was performed.

Fig. 3

Effect of ER store depletion on TPP-induced [Ca²⁺]_i. Fluo-3AM–labeled HL60 cells in Ca²⁺ free media were treated with a single concentration of TG before treatment with several equi-nicotine units of TPM (5, 10, and 20 μg/mL) or STE (100, 200, and 250 μg/mL). A single concentration of CaCl₂ was added to each concentration of TPP. Line graphs are from three independent experiments shown as mean ± SD. For each TPP, two-way analysis of variance was performed to test how [Ca²⁺]_i was affected by the presence or absence of CaCl₂. Post hoc analysis using Tukey’s honestly significant difference (HSD) test was performed and no adjusted p value was less than 0.05 for the relevant post hoc comparison.

Fig. 4

Effects of TRP channel inhibitors on TPM-induced calcium influx. Fluo-3AM–labeled cells were grown in Ca²⁺ free media with TRP channel inhibitor ruthenium red (Ru) or SKF-96365 (SK) and then treated with three concentrations of TPM (5, 10, and 20 μg/mL) followed by a single concentration of CaCl₂. DMSO was used as a negative vehicle control. Line graphs are from four independent experiments shown as mean ± SD.