NICOTINE EFFECTS ON NEUTROPHIL F-ACTIN FORMATION AND CALCIUM-RELEASE - IMPLICATIONS FOR TOBACCO USE AND PULMONARY-DISEASES

Alterations in neutrophil functions by tobacco components may play a p ivotal role in pulmonary emphysema. This study examined the role of ni cotine in altering F-actin formation and calcium (Ca2+) release (two e arly events in neutrophil motility. The effects of these alterations o n the motile function of phagocytosis were also examined. Human periph eral neutrophils from medically healthy nonsmoking subjects were incub ated with nicotine at concentrations normally encountered during acute exposure to cigarette smoke (10(-2) to 10(-5) M) and/or the chemotact ic peptide FLPEP (10(-7) M). Relative F-actin stain was determined by NBD phallacidin staining followed by flow cytometry. Intracellular Ca2 + was determined by INDO-1 AM loading followed by emission ratio quant itation by fluorometry. Phagocytosis was determined by the % phagocyti c cells with carboxylated microspheres. Incubation of neutrophils with varying concentrations of nicotine resulted in a significant elevatio n of the relative F-actin stain at 30 s at 10(-2) and 10(-3) M (p < .0 5, ANOVA) and at 30 min at 10(-2) to 10(-4) M (p < .05). In time cours e studies with 20(-7) M FLPEP stimulation, there was a similar to 325% rise in relative F-actin stain at 30-60 s, followed by a gradual decr ease to near baseline levels. There was an immediate rise in Ca2+ to s imilar to 150% over baseline values, followed by a gradual decrease to baseline. By contrast, stimulation with nicotine demonstrated a simil ar to 105% increase in relative F-actin staining at 10(-2) M (p < .001 , ANOVA) and a smaller increase at 10(-3) M, which remained elevated u p to 600 s. Intracellular Ca2+ levels also rose in a dose-dependent ma nner with an increase of 700% over baseline with 10(-2) M nicotine, an d remained elevated up to 600 s. Coincubation with both FLPEP and nico tine demonstrated additive effects in relative F-actin staining at bot h maximal and submaximal concentrations. Preincubation with 10(-2) or 10(-3) M nicotine suppressed the % phagocytic cells by 32% and 16%, re spectively (p < .001, ANOVA) with only a 1-4% reduction in cell viabil ity (trypan blue exclusion). The results demonstrate that the concentr ation of nicotine during acute cigarette exposure cart directly stimul ate netrtrophil F-actin formation and intracellular Ca2+ release by a mechanism different from peptide stimulation. The alteration of these two pivotal neutrophil signaling events by nicotine may in turn alter other neutrophil functions in tobacco-related pulmonary emphysema.