This is the first clear-cut demonstration that SHS increases the level of contractile ETA receptors in cerebral arteries via activation of the Raf/ERK/MAPK pathway. It is known that smokers or SHS exposure subjects have an increased risk to fall ill in stroke. However, the mechanisms behind this are poorly understood. Here, we show that the upregulation of ETA receptors with increased receptor-mediated vasoconstriction in the cerebral arteries observed after SHS exposure may be involved in SHS-related stroke. Specific inhibition of the Raf/ERK/MAPK pathway abolished the upregulation of ETA receptors in cerebral arteries of SHS exposed rats, while the other main MAPKs p38 and JNK were not affected.
Accumulating evidences indicate that both active and passive cigarette smoking are strongly associated with the origin and the development of stroke [1
]. There is a clear relation between smoking-related stroke risk, the dose-response relationship existence, and the costs of the smoke exposure on individuals and society [24
]. The present study was designed to imitate the manner of SHS exposure in man. It was found that animals required to be exposed to SHS for 8 weeks (200 min/day of passive smoke from the lit cigarette) to show ETA
receptor changes. Two or 4 weeks of SHS did not alter ET receptor-mediated vasoconstriction in cerebral arteries. After 8 weeks of SHS exposure there was a significant increase in cerebral artery contraction mediated by ETA
receptors. Basically, enhanced cerebral vasoconstriction mediated by receptors can be attributed to upregulated (presence of more) receptors and/or increased sensitivity of cerebral vessels in response to receptor agonist [22
]. Since the contractile response mediated by receptors is considered a reflection of receptor expression in cerebral arteries, the receptor-mediated vasoconstriction is in accord with enhanced receptor levels. In agreement, results of mRNA and protein expressions of ETA
receptors were in support of our hypothesis of more receptors. These results reveal that SHS upregulates the ETA
a transcriptional mechanism. SHS exposure did not alter ETB
receptor expression or the receptor-mediated contraction. This implies the method to culture cerebral arteries with tobacco extracts in vitro
] differs from passive smoke exposure in the whole animal in vivo
. Furthermore, SHS did not alter the K+
induced contraction in any group which further suggests specificity in the receptor upregulation process.
It is known that the ET-1 levels in blood and CSF are increased in stroke; this may be further translated to an enhanced receptor-mediated contraction in cerebral arteries [26
]. Transcriptional upregulation of ETA
receptors has been reported in rat cerebral arteries after using some injury models like experimental cerebral ischemia and organ culture [10
]. In all cases, the receptor upregulation occurred in the smooth muscle cells. The similar findings were confirmed in cerebral vessels from ischemic stroke patients [29
]. Consequently, we believe that the ETA
receptor was also increased in smooth muscle cells in the present study. Currently it reveal that SHS induces enhanced expression of ETA
receptor mRNA and protein in cerebral arteries; this implies an important role in SHS-associated stroke. The significance remains to be tested in SHS-exposed animals using experimental stroke models; possibly they may show larger infarcts after an experimental stroke.
MAPKs have an important role in cerebrovascular receptor plasticity [3
]. Particularly for ERK1/2, it located downstream of a dynamic chain of the kinases and is considered mainly mitogenic and has a predominant role in growth factor receptor signaling. We have demonstrated activation of ERK1/2 in cerebral arteries after MCAO and cerebral ischemia [18
]. On this basis, the involvement of ERK1/2 pathway was assessed in the contractile receptor upregulation in artery culture [27
]. Recently, a number of MAPK inhibitors were used to compare their ability to prevent the upregulation of various cerebrovascular vasoconstrictor receptors during organ culture [14
]. In the present study we demonstrated SHS exposure induced ERK1/2 signaling activation by enhanced ERK1/2 phosphorylation. Moreover, we showed that SHS upregulated ETA
receptors in rat cerebral arteries. It means SHS-induced ETA
upregulation occurs through ERK1/2 activation. Meanwhile, we used a Raf-1 inhibitor GW5074 and confirmed that it is Raf/ERK1/2 signaling involved in the SHS-induced receptor changes, but not JNK or p38 pathway. This hypothesis is also supported by our recent in vitro
discovery in cerebral arteries exposed to lipid-soluble smoke particles [28
Raf-1 is associated ubiquitously in the Raf/MEK/ERK pathway. Raf phosphorylates MEK1/2, which in turn phosphorylates and activates ERK1/2 and then leads to activation of transcription factors [32
]. The ERK1/2 pathway is a major effector of Raf. Transient activation of Raf-1 contributes to alterations in smooth muscle cell function, such as enhanced contraction and proliferation, whereas sustained activation results in differentiation via
the regulation of various ERK substances [33
]. We chose the Raf-1 inhibitor GW5074 to further demonstrate the involvement of ERK in the ET receptor upregulation after SHS. The specificity and efficacy of GW5074 for inhibiting Raf-1 in-vivo
has been established in previous studies [32
]. Lakey et al.
and Chin et al.
reported that GW5074 is a potent Raf-1 inhibitor and examined the effect of GW5074 on purified Raf-1 and confirmed that GW5074 selectively inhibits Raf-1 in vivo
. In the present study, GW5074 attenuated the SHS-induced elevated cerebral artery contraction as well as increased mRNA mediated by ETA
receptors. This strongly supports that SHS induces ETA
receptor upregulation via
the Raf/ERK/MAPK pathway. We demonstrated the mRNA of Raf-1 and ERK1/2 was increased after SHS, but the total Raf-1 or ERK1/2 proteins were not changed. We think the former measurement reflects steady state and thus that may also be other changes such as in degradation or mRNA stability.
The enhanced phosphorylation of Raf-1 and ERK1/2 suggests the Raf/ERK1/2 pathway has been activated. The kinases elicit some of their effects through phosphorylation of transcriptional regulation. Currently, Raf-1 inhibitor GW5074 reduced phosphorylation of ERK1/2 as well as Raf-1. The GW5074-induced declined phosphorylation of ERK1/2 should be attributed to the upstream inhibition of ERK1/2. However, the reason to explain the reduced Raf-1 phosphorylation is not sure. We think it may be some upstream influences or feedback mechanisms when blocking Raf-1 activity by GW5074. It could be a partial reason for decreased Raf-1 phosphorylation. Moreover, we performed in vivo treatment in the animals. It may also be some possible indirect effects of GW5074 that altered Raf-1 phosphorylation when administrated with the inhibitor in vivo. However, the overall data agree with the involvement of Raf/ERK/MAPK in SHS.