Secondhand smoke exposure induces Raf/ERK/MAPK-mediated upregulation of cerebrovascular endothelin ETA receptors

Abstract

Background: Cigarette smoking enhances the risk of stroke. However, the underlying molecular mechanisms are largely unknown. The present study established an in vivo rat secondhand cigarette smoking (SHS) model and examined the hypothesis that SHS upregulates endothelin receptors with increased cerebrovascular contraction via the Raf/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK) pathway.

Results: Rats were exposed to SHS for up to 8 weeks. The cerebral artery vasoconstriction was recorded by a sensitive myograph. The mRNA and protein expressions for endothelin receptors in cerebral arteries were studied by real-time PCR and Western blot. Compared to fresh air exposed rats, cerebral arteries from SHS rats exhibited stronger contractile responses (P < 0.05) mediated by endothelin type A (ETA) receptors. The expressions of mRNA and protein for ETA receptors in the cerebral arteries from SHS rats were higher (P < 0.05) than that in control. SHS did not affect endothelin type B (ETB) receptor-mediated contractions, mRNA or protein levels. The results suggest that SHS upregulates ETA, but not ETB receptors in vivo. After SHS exposure, the mRNA levels of Raf-1 and ERK1/2, the protein expression of phosphorylated (p)-Raf-1 and p-ERK1/2 were increased (P < 0.05). Raf-1 inhibitor, GW5074 suppressed the enhanced ETA receptor-mediated contraction, mRNA and protein levels induced by SHS. In addition, GW5074 inhibited the SHS-caused increased mRNA and phosphorylated protein levels of Raf-1 and ERK1/2, suggesting that SHS induces activation of the Raf/ERK/MAPK pathway.

Conclusions: SHS upregulates cerebrovascular ETA receptors via the Raf/ERK/MAPK pathway, which provides novel understanding of mechanisms involved in SHS-associated stroke.

Figure 1

Contractile responses induced by S6c (A) and ET-1 (B) in rat cerebral arteries. The cerebral arteries were isolated from groups of fresh air, SHS and SHS treatment with GW5074. Contractions were induced by cumulative application of S6c or ET-1 (n = 8-10). *P < 0.05, **P < 0.01 versus fresh air group, ^#P < 0.05 versus SHS group; SHS, secondhand smoke exposure; GW, GW5074 (0.5 mg/kg).

Figure 2

The levels of ET_B and ET_A receptor mRNA (A) and protein (B) expression in rat cerebral arteries. The cerebral arteries were isolated from groups of fresh air, SHS and SHS treatment with GW5074. Receptor mRNA content was examined by real-time PCR (n = 5-8) and the protein level was examined by Western blot (n = 5-6). *P < 0.05, **P < 0.01 versus fresh air group, ^#P < 0.05 versus SHS group; SHS, secondhand smoke exposure; GW, GW5074 (0.5 mg/kg).

Figure 3

The level of MAPK molecule (ERK1, ERK2, p38α and JNK1) mRNA expression in rat cerebral arteries (A). Activation of Raf-1 protein expression in rat cerebral arteries (B). Activation of ERK1/2 protein expression in rat cerebral arteries (C). The cerebral arteries were isolated from groups of fresh air, SHS and SHS treatment with GW5074. The key MAP kinases mRNA was relative to GAPDH (n = 5-6); and the p-Raf-1 and p-ERK1/2 protein were relative to Raf-1 or ERK1/2 level (n = 5). *P < 0.05, **P < 0.01 versus fresh air group, ^#P < 0.05 versus SHS group, ns not significant; SHS, secondhand smoke exposure; GW, GW5074 (0.5 mg/kg).