Alteration in contractile G-protein coupled receptor expression by moist snuff and nicotine in rat cerebral arteries

Abstract

The cardiovascular risk for users of use of Swedish snus/American snuff (moist tobacco) has been debated for a long time. The present study was designed to examine the effects of water- or lipid-soluble (DMSO-soluble) snus and nicotine, the most important substance in tobacco, on the expression of vasocontractile G-protein coupled receptors (GPCR), such as endothelin ET(B), serotonin 5-HT(1B), and thromboxane A(2) TP receptors, in rat cerebral arteries. Studies show that these vasocontractile GPCR show alterations by lipid-soluble cigarette smoke particles via activation of mitogen-activated protein kinases (MAPK). However, the effects of moist tobacco on the expression of GPCR are less studied. Rat middle cerebral arteries were isolated and organ cultured in serum-free medium for 24h in the presence of water-soluble snus (WSS), DMSO-soluble snus (DSS), or nicotine. The dose of snus and nicotine was kept at plasma level of snus users (25ng nicotine/ml). A high dose (250ng nicotine/ml) was also included due to the previous results showing alteration in the GPCR expression by nicotine at this concentration. Contractile responses to the ET(B) receptor agonist sarafotoxin 6c, 5-HT(1B) receptor agonist 5-carboxamidotryptamine, and TP receptor agonist U46619 were investigated by a sensitive myograph. The expression of ET(B), 5-HT(1B), and TP receptors was studied at mRNA and protein levels using quantitative real-time PCR and immunohistochemistry, respectively. Organ culture with WSS or DSS (25ng nicotine/ml) lowered the 5-HT(1B) receptor-mediated contraction. Furthermore, DSS shifted the TP receptor-mediated contraction curve left-wards with a stronger contraction. High dose of nicotine (250ng nicotine/ml) increased the ET(B) receptor-mediated contraction. The combined 5-HT(1B) and 5-HT(2A) receptor-mediated contraction was increased, and both the 5-CT and TxA2 induced contractions were left-ward shifted by WSS, DSS, or nicotine (250ng nicotine/ml). Only the DSS group showed that the increase of 5-HT(1B) receptor-mediated contraction occurred at the transcriptional level, demonstrated by an increased mRNA expression for the receptor. Thus, snus and nicotine alter the GPCR expression in the cerebral arteries, which may be involved in cerebral vascular disease.