Lipid‐soluble Cigarette Smoke Particles Induced Vascular Endothelin Type A Receptor Up‐Regulation through Activation of ERK1/2 Signal Pathways

Abstract

Abnormal contraction of vessels termed 'vasospasm' is associated with various cardiovascular diseases. Smoking is a well-known risk factor that increases vasospasm. However, the molecular mechanisms by which smoking leads to vasospasm and cardiovascular disease are not fully understood. This study was designed to examine whether DMSO-extracted cigarette smoke particles (DSP) could induce up-regulation of vascular endothelin type A (ET ) receptors, and whether ET receptor is up-regulated through activation of extracellular regulated protein kinases 1 and 2 (ERK1/2) signal pathways. Mesenteric arterial segments from rats were cultured in the presence of DSP, water-extracted cigarette smoke particles (WSP) or equivalent concentration of nicotine for up to 24 hr. The results showed that DSP, but not WSP or nicotine, induced ET receptor up-regulation with increased ET receptor-mediated contraction (myograph, p < 0.001). Simultaneously, the expression of ET receptor mRNA (real-time PCR, p < 0.001) and protein (immunohistochemistry) were enhanced in the smooth muscle cells, suggesting that the lipid-soluble substances contained in cigarette smoke were responsible for the effects of DSP. Actinomycin D (a general transcriptional inhibitor) decreased ET receptor mRNA expression and attenuated receptor-mediated contraction (p < 0.001), while DSP accelerated ET receptor mRNA degradation (p < 0.01) and promoted the translation of ET receptor mRNA into protein. Furthermore, the up-regulation of ET receptors was significantly attenuated by inhibition of ERK1/2 signal pathways (p < 0.001). In conclusion, DSP most likely activate ERK1/2 signal pathway-mediated transcriptional and post-transcriptional (translational) mechanisms that lead to vascular ET receptor up-regulation, which might contribute to vasospasm and the development of smoking-associated cardiovascular diseases.

Enhanced Endothelin A and B Receptor Expression and Receptor-Mediated Vasoconstriction in Rat Mesenteric arteries after Lipopolysaccharide Challenge

“…To understand the association between ETR alteration and cardiovascular risk factors, for instance, cigarette smoking and dyslipidemia, we established an in vitro organ culture model and studied the effects of smoking particle extracts and LDL. It is intriguing to discover that ET A and ET B2 expressions were upregulated by exposure to dimethyl sulfate-soluble smoking particles [6, 7], or to native LDL (through lipid peroxidation) [11]. These findings were supported by the results published by several groups [22, 23].…”

“…The mesenteric tissue sample was gently removed from the abdomen. Dissection of the mesenteric artery and denudation of the endothelium with Triton X-100 were performed as described previously [6]. All the animal experimental procedures were approved by the Ethics Committee on Animal Research from The First Affiliated Hospital of Xiamen University, complying with Animal Research: Reporting of In Vivo Experiments (ARRIVE) Guidelines, and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th Edition) and American Veterinary Medical Association (AMVA) Guidelines for the Euthanasia of Animals (2013 Edition).…”

“…The arteries (without endothelium) were cut into 1 mm long cylindrical segments and mounted to a myograph system (620 M, Danish Myo Technology A/S, Aarhus, Denmark) for recording the receptor-mediated vasoconstriction. The concentration-response curves (CRCs) were performed by cumulative administration of selective ET B agonist sarafotoxin 6c (S6c, Sigma-Aldrich, Saint Louis, MO, USA), followed by nonselective ETR agonist ET-1 (Calbiochem, La Jolla, CA, USA), as previously described [6, 11]. Briefly, after S6c CRCs were obtained, the arterial rings were coincubated with S6c (10 −7.5 M) for 30 min.…”

“…Real-time quantitative PCR was performed in a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) with the reaction protocol as described previously [6]. Primers were as follows: ET A (Ednra, GenBank accession no.…”

“…Previously, by using an in vitro organ culture model, we demonstrated the upregulation of ET B2 expression in VSMC through the activation of the nuclear factor- κ B (NF- κ B) pathway after exposure of the arteries to tumor necrosis factor- α (TNF- α ), a key proinflammatory cytokine [5]. Additionally, during organ culture, expressions of ET A and ET B2 were enhanced by lipid-soluble smoking particles [6, 7] and low-density lipoprotein (LDL) [8]. The above data, to some aspect, indicated a link between upregulation of ETRs and inflammation associated with cardiovascular risk factors.…”