Nicotine Delays Bone Growth Figure 4. Calcium influx assay in primary chondrocyte culture. Nicotine-stimulated calcium signaling was investigated by the use of a fluorescent Ca2+ indicator. Primary chondrocyte cultures were stimulated by nicotine with or without MLA, the specific antagonist of alpha7 homomeric nAChR. A: Addition of assay buffer alone elicits no reaction (upper panels: negative control). Nicotine elicits a transient increase of intra- cellular calcium (lower panels). B: Nicotine elicits a transient increase of intra-cellular calcium in a concentration-dependent manner. C: MLA inhibits nicotine-induced calcium influx in a concentration-dependent manner. The cells were treated with MLA 30 min before nicotine stimulation. doi:10.1371/journal.pone.0003945.g004 (Figure 6C). Besides, scatterplot and correlation between the FL and the BW revealed that nicotine downwardly shifted the linear slope in alpha7 nAChR +/+ fetuses but had no effect in alpha7 nAChR 2/2 fetuses (Figure 6D). These findings suggest that maternal nicotine exposure decreased the fetal endochondral ossification through the fetal alpha7 nAChR in vivo. Discussion Alpha7 nAChR was originally identified as a subunit of neuronal nAChR, and has also been shown to be functional in both neuronal and non-neuronal, i.e., non-excitable cells such as lymphocytes, vascular endothelial cells, keratinocytes and bron- chial epithelium [16]. In this study, we demonstrated the expression of the alpha7 subunit of nAChR at resting to pre- hypertrophic chondrocytes in murine growth plate and on a culture of human growth plate chondrocytes, and the involvement of alpha7 nAChR in nicotine-induced delayed skeletal growth. The novel findings of alpha7 nAChR in chondrocytes suggest that the effect of smoking on delayed skeletal growth is directly correlated with nicotinic action on chondrocytes. Direct effect of nicotine on human growth plate chondrocytes Maternal nicotine exposure decreases the width of the hypertrophic zone of growth plate, increases apoptotic chondro- cytes, and reduces the length of femur in rat [20]. Contrarily, nicotine has been shown to up-regulate glycosaminoglycan and PLoS ONE | www.plosone.org 6 December 2008 | Volume 3 | Issue 12 | e3945