Nicotine and polycyclic aromatic hydrocarbons modulate junctional proteins and paracellular permeability of rat cerebral endothelial cells

Abstract

Cigarette smoking contributes to the development of several neurological disorders associated with blood-brain barrier (BBB) dysfunction. However, very little is know about the effect of smoking on the BBB. Our work was aimed on the effect of major cigarette smoke components on rat cerebral endothlial cells (CECs). Western blot analysis was used to investigate whether nicotine and polycyclic aromatic hydrocarbons (PAH), cigarette smoke components, had an effect on junctional protein expression. The results revealed that the rat CECs exposed to 10 muM nicotine 24 h significantly decreased cadherin and ZO-1 protien expression in triton X-100 (Tx-100) soluble fraction while no change was observed either in the case of phenanthrene or 1-methylanthracene exposure at the concentration of 30muM. Immunostaining experiments were used to determine the possible rearrangment in the subcellular distribution of cadherin and ZO-1. A disruption in the continuity of cadherin and ZO-1 staining along cell-cell contacts were observed, which was correlated with the observation obtained by Weatern blot analysis. No change of protein distribution after the treatment with either phenanthrene or 1-methylanthracene was observed. Due to the observation of a significant change in ZO-1 protein expression after nicotine exposure, protein-protein interactions within the rat CECs were further investigated using co-immunoprecipitation assay. The results showed that protein-protein interactions between occludin and ZO-1 decreased after nicotine exposure. To assess the potential damaging effect of cigarette smoke components on the barrier function, transendothelial electrical resistance (TEER) was measured in a coculture model using rat CECs and astrocytes. Although nicotine decreased ZO-1 protein expression and localization of junctional proteins, the barrier permeability evaluated by TEER measurements did not change in our study. The barrier permeability was also not affected by the treatment with either phenanthrene or 1-methylanthracene. There are several reports showing that oxidative stress is implicated in pathological processes of the central nervous system. Therefore, the potential cumulative effect of oxidative stress and nicotine on the BBB permeability was investigated. Immunostaining analysis revealed that co-treatment of the rat CECs with 10 muM nicotine in the presence of 10 muM 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) disrupted the continuity of ZO-1 staining along the cell-cell contacts, which was more pronounce compared to DMNQ or nicotine treatmemt alone. Our results revealed that nicotine treatmemt under oxidative stress for 24 h increased the transendothelial permesbility. In conclusion, nicotine significantly affects the junctional complex of the rat CECs by changing protein expression and protein localization of cadherin and ZO-1. The damaging effects are enhanced by oxidative stress, which can further disturb the function of the BBB.