Nicotine effects on human endothelial intercellular communication via α4β2 and α3β2 nicotinic acetylcholine receptor subtypes

Since previous in vitro experiments revealed that nicotine can impair endothelial intercellular communication via the downregulation of connexin43 (Cx43), we wanted to find out which nicotinic acetylcholine receptors are involved in the molecular mechanism of communication failure. Cultured human endothelial cells were exposed to 1 mu M nicotine for 5 days. Intercellular communication was measured using dye transfer study with/without subtype-specific nicotinic acetylcholine receptor (nAChR) inhibitors. Reverse transcriptase (RT)-PCR was used to further investigate the regulation of nAChR subtypes. Electron microscopy together with MAP LC3-II western blot was used to investigate possible autophagy processes. In cultured human endothelial cells, nicotine decreased the Cx43 protein amount as shown by western blot and immunohistochemistry; however, together with an unaltered mRNA expression as shown by RT-PCR. The nicotine-induced Cx43 downregulation functionally impaired intercellular dye transfer, which could be prevented by mecamylamine, kappa-bungarotoxin, lobeline, and dihydro-beta-erythroidine but not alpha-bungarotoxin, indicating that the nAChR subtypes alpha 4 beta 2 and alpha 3 beta 2 but not alpha 7 are involved in signal cascade. RT-PCR analysis revealed that nicotine exposure resulted in the upregulation of alpha 3 and beta 4 and the downregulation of alpha 4-nAChR, while alpha 7- and beta 2-nAChR-mRNA expressions remained unaltered. Furthermore, nicotine increased total protein ubiquinylation and proteasome activity as was shown by immunohistochemistry and peptide degradation analysis. Evidence of enhanced autophagic processes was assured by the occurrence of autophagic vacuoles in transmission electron microscopy and enhanced formation of MAP LC3-II in western blot. Reduced intercellular endothelial communication together with programmed cell death helps to explain the toxic effect of nicotine leading to endothelial dysfunction. The nAChR involved in the impairment of intercellular communication seem to be alpha 4 beta 2 and alpha 3 beta 2 but not alpha 7.