BACKGROUND
passive smoke exposure or secondhand smoke  is strongly associated with ischemic and hemorrhagic stroke  <cit> , and has harmful effects on the structure and function of cerebral blood vessels, promoting atherosclerosis and stiffening of arteries  <cit> .
however, the biological basis of shs on the vessel walls is not well understood.
endothelin - <dig> is one of the most potent vasoconstriction found in the circulation with elevated levels in stroke  <cit> .
et- <dig> is produced by endothelial cells, mediates its vasomotor response through two different g-protein coupled receptors, the endothelin type a  and the endothelin type b  receptor  <cit> .
in cerebral vessels, the eta receptors are found mainly on the smooth muscle cells and mediate strong vasoconstriction  <cit> , while etb receptors are primarily situated on the endothelium of cerebral vessels and stimulate the formation of nitric oxide and prostacyclin mediating vasodilatation  <cit> .
because et- <dig> causes potent and long-lasting vasoconstriction, and there are increased levels of et- <dig> in cerebral spinal fluid  after subarachnoid hemorrhage   <cit> ; it has been suggested to play an important role in the pathogenesis of delayed cerebral vasospasm following sah  <cit>  and in cerebral ischemia  <cit> .
in addition, there are increasing evidences demonstrating that experimental sah and cerebral ischemia may be associated with et receptor upregulation in cerebral artery smooth muscle cells  <cit> .
the main risk factors for stroke in general include hyperlipidemia, hypertension and cigarette smoking  <cit> .
here we address in particular one of these, shs, which is associated with increased risk of sah and ischemic stroke in general population  <cit> .
our previous in vitro studies have demonstrated that lipid-soluble smoke particles, but not water soluble smoke particles or nicotine per se, induce etb receptor upregulation in cerebral vessels  <cit> .
the increased receptors result in enhanced contractility and local inflammation.
to the best of our knowledge, it has not been studied if shs in vivo is associated with elevated expression of et receptors.
if both the formation of et- <dig> and the number of contractile et receptors are increased in individuals after exposure to shs, it may bring about larger damage in sah or cerebral infarct, compared to the non-smokers.
we hypothesize that shs exposure in vivo upregulates et receptors in cerebral arteries, which may in turn contribute to larger brain damage in stroke among smoke exposed subjects.
the cellular mechanisms involved in shs-associated stroke are unclear; here we examine if the et receptor upregulation induced by shs is associated with intracellular mitogen-activated protein kinase  signaling.
this system consists of extracellular signal-regulated protein kinase  <dig> and  <dig> , c-jun n-terminal kinase  and p <dig> pathways.
raf- <dig> is the initial protein kinase in the mapk signal transduction pathway which phosphorylates subsequent map kinase/extracellular signal-regulated kinase kinase  <dig> and  <dig>   <cit> .
we have recently in detail described that activation of mapk-mediated signal transduction is associated with upregulation of et receptors in cerebral vasculature and that et receptor expression is enhanced in ischemic stroke  <cit> .
the importance of mapk signaling in the pathophysiology of ischemic stroke has been widely studied.
increased erk1/ <dig> phosphorylation has been observed in the ischemic area after both transient and permanent middle cerebral occlusion, as well as after global ischemia  <cit> .
consequently, inhibitors of erk1/ <dig> and mek1/ <dig> have been effective in reducing the infarct size in cerebral ischemia  <cit> , and in sah  <cit> .
erk1/ <dig> is also activated in the cerebral arteries of the ischemic brain, pointing towards a role in vascular alterations  <cit> .
however, it is not known if the risk factor shs per se may alter et receptor expression in cerebral arteries and if this is associated with intracellular signaling via the raf/erk/mapk pathway.
the present study was designed, using an in vivo rat passive smoke exposure model, to demonstrate that cigarette smoke may upregulate cerebrovascular et receptors, and to examine the intracellular signal mechanisms of shs-induced enhanced et receptor expression by in vivo treatment with a specific raf- <dig> inhibitor.
RESULTS
general
there was no significant difference in cerebral artery contractile responses to k+, sarafotoxin  <dig> c  and et- <dig> after  <dig> or  <dig> weeks in shs exposed rats as compared to rats exposed to fresh air for a similar time period .
therefore, we only present detailed results from the  <dig> weeks of exposure to shs.
the cerebral arteries were from groups of fresh air and shs for  <dig> and  <dig> weeks.
k+-induced responses were expressed as absolute values of contraction .
s6c and et-1-induced responses were expressed as percentage of k+-induced contraction.
emax, maximal contraction; shs, secondhand smoke exposure.
effects of shs on et receptor-mediated contractions in cerebral artery
the contraction elicited by k+ was used as a reference for the contractile capacity.
k+- induced contractile responses did not differ significantly in artery segments from fresh air, shs and shs plus inhibitor groups .
the etb receptor-mediated contraction was examined using the specific etb receptor agonist s6c, which has been characterized in detail before using the etb receptor antagonist irl <dig>  <cit> .
the vasoconstriction induced by a combined eta and etb receptor agonist et- <dig> was studied after desensitizing the etb receptors with s6c prior to adding et- <dig>  leaving only eta receptors to respond.
this has been verified by use of the selective eta receptor antagonist fr <dig>  <cit> .
the cerebral arteries were from groups of fresh air, shs and shs treatment with gw <dig> for  <dig> weeks.
k+-induced responses were expressed as absolute values of contraction .
s6c and et-1-induced responses were expressed as percentage of k+-induced contraction.
*p <  <dig>  versus fresh air group.
emax, maximal contraction; pec <dig>  negative logarithm of the concentration that produces 50% of the maximal contractile effect; shs, secondhand smoke exposure; gw, gw <dig> .
s6c only induced a slight contraction  in cerebral arteries of fresh air exposed rats.
there was no significant difference in s6c-induced contractions between shs and fresh air groups .
after desensitization of etb receptors, cumulative administration of et- <dig> induced potent contraction of fresh arteries in a concentration-dependent manner  with an emax of  <dig> ± 9%.
following shs the concentration-response curve showed an increased emax  with no significant difference in pec <dig> values .
this indicates that the efficacy of the response is increased after shs exposure.
effects of shs on et receptor mrna and protein expressions
the mrna and protein levels of etb and eta receptors in cerebral arteries were measured by real-time pcr and western blot, respectively.
the standard curves of each primer pair in the qpcr had almost similar slopes, indicating that gapdh and receptor cdnas were amplified with the same efficiency   <cit> .
the values of each slope were close to  <dig> , meaning that the amplification efficiencies were almost optimal.
there was no significant contaminating nucleic acid in blank control samples.
the etb receptor mrna expression remained unaltered after shs exposure as compared to control .
the protein level of etb receptor relative to β-actin was  <dig>  ±  <dig>  in fresh air exposed rats, and  <dig>  ±  <dig>  in the shs exposed group .
these results were in concert with the functional myograph studies.
the mrna level for the eta receptor relative to gapdh was significantly elevated after shs in cerebral arteries .
the level of eta receptor protein was  <dig>  ±  <dig>  relative to β-actin in the fresh air group and enhanced to  <dig>  ±  <dig>  after shs .
taken together, the results demonstrate that shs induces eta receptor upregulation.
mapk signal pathway studies
to investigate the underlying intracellular signal transduction mechanisms associated with the shs-induced increase in eta receptor expression, we first examined the mrna levels of several key protein kinases such as raf- <dig>  erk <dig>  erk <dig>  p38α and jnk <dig> by real-time pcr.
the results showed that shs increased mrna levels of raf- <dig>  erk <dig> and erk <dig> as compared to the fresh air group.
shs had no effect on p38α and jnk <dig> mrna levels .
in addition, we examined the phosphorylated -raf- <dig>  p-erk1/ <dig>  p-p <dig> and p-jnk, and their total protein expressions by western blot in rat cerebral vessels.
total raf- <dig> and erk1/ <dig> protein were unaltered in the shs group compared to fresh air group.
the phosphorylated protein of raf- <dig> was enhanced to  <dig>  ±  <dig>   from  <dig>  ±  <dig>  .
the level of p-erk1/ <dig> protein relative to erk1/ <dig> in fresh air and shs animals were  <dig>  ±  <dig>  and  <dig>  ±  <dig>  , respectively .
in contrast, the protein level of p-p <dig> was  <dig>  ±  <dig>   and  <dig>  ±  <dig>  ; the level of p-jnk protein was  <dig>  ±  <dig>   and  <dig>  ±  <dig>  .
we did not find any significant difference in protein expression of p-p <dig> and p-jnk between fresh air and shs groups.
the results show that shs induces enhanced expression of cerebrovascular eta receptors via the raf/erk1/ <dig> activation, but does not appear to involve jnk or p <dig> pathways in the present experimental conditions.
effect of gw <dig> on shs-induced effects
in order to further understand the role of raf/erk/mapk signal pathway, we studied the inhibitory effect of daily gw <dig> administration on shs-induced responses.
the results showed that the raf- <dig> and erk <dig> mrna levels in cerebral arteries were significantly lower after gw <dig> treatment in shs exposed animals .
erk <dig> mrna showed a somewhat lower level after inhibition which was not different from the shs group .
the mrna levels of other protein kinases  remained unaltered by gw <dig> treatment in shs.
western blotting confirmed the mrna results.
there was a significant decrease in the p-raf- <dig> and p-erk1/ <dig> protein level after gw <dig> treatment as compared with that of shs .
the p-p <dig> and p-jnk proteins remained unchanged between the smoke exposure and treatment groups.
the protein level of p-p <dig> was  <dig>  ±  <dig>   and  <dig>  ±  <dig>  ; the level of p-jnk protein was  <dig>  ±  <dig>   and  <dig>  ±  <dig>  , respectively.
shs or shs with the raf- <dig> inhibitor did not modify the weak s6c-induced contraction after  <dig> weeks of shs combined with daily administration of gw <dig> .
in contrast, treatment with gw <dig> markedly attenuated shs-induced enhanced cerebral vasoconstriction elicited by et- <dig> at eta receptors .
the emax of the et- <dig> induced concentration-contractile curve in gw <dig> treatment group was now the same as that seen in rats exposed to fresh air for  <dig> weeks .
the mrna and protein levels of et receptors were also determined after treatment with raf- <dig> inhibitor.
the etb receptor mrna and protein levels were unchanged in the inhibitor group as compared to shs or fresh air groups .
the mrna level of eta receptors was significantly reduced after inhibition of raf/erk/mapk in the shs group .
the protein level of eta receptor was lower in the gw <dig> treatment group but did not reach statistical significance .
discussion
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 p <dig> 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  <cit> .
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  <cit> .
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  <dig> weeks  to show eta receptor changes.
two or  <dig> weeks of shs did not alter et receptor-mediated vasoconstriction in cerebral arteries.
after  <dig> 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  receptors and/or increased sensitivity of cerebral vessels in response to receptor agonist  <cit> .
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 receptor via 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  <cit>  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- <dig> levels in blood and csf are increased in stroke; this may be further translated to an enhanced receptor-mediated contraction in cerebral arteries  <cit> .
transcriptional upregulation of eta and etb receptors has been reported in rat cerebral arteries after using some injury models like experimental cerebral ischemia and organ culture  <cit> .
in all cases, the receptor upregulation occurred in the smooth muscle cells.
the similar findings were confirmed in cerebral vessels from ischemic stroke patients  <cit> .
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  <cit> .
particularly for erk1/ <dig>  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/ <dig> in cerebral arteries after mcao and cerebral ischemia  <cit> .
on this basis, the involvement of erk1/ <dig> pathway was assessed in the contractile receptor upregulation in artery culture  <cit> .
recently, a number of mapk inhibitors were used to compare their ability to prevent the upregulation of various cerebrovascular vasoconstrictor receptors during organ culture  <cit> .
in the present study we demonstrated shs exposure induced erk1/ <dig> signaling activation by enhanced erk1/ <dig> phosphorylation.
moreover, we showed that shs upregulated eta receptors in rat cerebral arteries.
it means shs-induced eta upregulation occurs through erk1/ <dig> activation.
meanwhile, we used a raf- <dig> inhibitor gw <dig> and confirmed that it is raf/erk1/ <dig> signaling involved in the shs-induced receptor changes, but not jnk or p <dig> pathway.
this hypothesis is also supported by our recent in vitro discovery in cerebral arteries exposed to lipid-soluble smoke particles  <cit> .
raf- <dig> is associated ubiquitously in the raf/mek/erk pathway.
raf phosphorylates mek1/ <dig>  which in turn phosphorylates and activates erk1/ <dig> and then leads to activation of transcription factors  <cit> .
the erk1/ <dig> pathway is a major effector of raf.
transient activation of raf- <dig> 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  <cit> .
we chose the raf- <dig> inhibitor gw <dig> to further demonstrate the involvement of erk in the et receptor upregulation after shs.
the specificity and efficacy of gw <dig> for inhibiting raf- <dig> in-vivo has been established in previous studies  <cit> .
lakey et al.
and chin et al.
reported that gw <dig> is a potent raf- <dig> inhibitor and examined the effect of gw <dig> on purified raf- <dig> and confirmed that gw <dig> selectively inhibits raf- <dig> in vivo.
in the present study, gw <dig> 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- <dig> and erk1/ <dig> was increased after shs, but the total raf- <dig> or erk1/ <dig> 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- <dig> and erk1/ <dig> suggests the raf/erk1/ <dig> pathway has been activated.
the kinases elicit some of their effects through phosphorylation of transcriptional regulation.
currently, raf- <dig> inhibitor gw <dig> reduced phosphorylation of erk1/ <dig> as well as raf- <dig>  the gw5074-induced declined phosphorylation of erk1/ <dig> should be attributed to the upstream inhibition of erk1/ <dig>  however, the reason to explain the reduced raf- <dig> phosphorylation is not sure.
we think it may be some upstream influences or feedback mechanisms when blocking raf- <dig> activity by gw <dig>  it could be a partial reason for decreased raf- <dig> phosphorylation.
moreover, we performed in vivo treatment in the animals.
it may also be some possible indirect effects of gw <dig> that altered raf- <dig> phosphorylation when administrated with the inhibitor in vivo.
however, the overall data agree with the involvement of raf/erk/mapk in shs.
CONCLUSIONS
the present study is the first to show that passive smoke exposure upregulates eta, but not etb receptors, in rat cerebral arteries.
the upregulation of eta receptors occurs via activation of the raf/erk/mapk pathway.
this mechanism may provide new options for treatment of shs-associated cerebrovascular diseases.

 passive smoke exposure or secondhand smoke is strongly associated with ischemic and hemorrhagic stroke, and has harmful effects on the structure and function of cerebral blood vessels, promoting atherosclerosis and stiffening of arteries. however, the biological basis of shs on the vessel walls is not well understood. endothelin - is one of the most potent vasoconstriction found in the circulation with elevated levels in stroke. et- is produced by endothelial cells, mediates its vasomotor response through two different g-protein coupled receptors, the endothelin type a and the endothelin type b receptor. in cerebral vessels, the eta receptors are found mainly on the smooth muscle cells and mediate strong vasoconstriction, while etb receptors are primarily situated on the endothelium of cerebral vessels and stimulate the formation of nitric oxide and prostacyclin mediating vasodilatation. because et- causes potent and long-lasting vasoconstriction, and there are increased levels of et- in cerebral spinal fluid after subarachnoid hemorrhage; it has been suggested to play an important role in the pathogenesis of delayed cerebral vasospasm following sah and in cerebral ischemia. in addition, there are increasing evidences demonstrating that experimental sah and cerebral ischemia may be associated with et receptor upregulation in cerebral artery smooth muscle cells. the main risk factors for stroke in general include hyperlipidemia, hypertension and cigarette smoking. here we address in particular one of these, shs, which is associated with increased risk of sah and ischemic stroke in general population. our previous in vitro studies have demonstrated that lipid-soluble smoke particles, but not water soluble smoke particles or nicotine per se, induce etb receptor upregulation in cerebral vessels. the increased receptors result in enhanced contractility and local inflammation. to the best of our knowledge, it has not been studied if shs in vivo is associated with elevated expression of et receptors. if both the formation of et- and the number of contractile et receptors are increased in individuals after exposure to shs, it may bring about larger damage in sah or cerebral infarct, compared to the non-smokers. we hypothesize that shs exposure in vivo upregulates et receptors in cerebral arteries, which may in turn contribute to larger brain damage in stroke among smoke exposed subjects. the cellular mechanisms involved in shs-associated stroke are unclear; here we examine if the et receptor upregulation induced by shs is associated with intracellular mitogen-activated protein kinase signaling. this system consists of extracellular signal-regulated protein kinase and, c-jun n-terminal kinase and p pathways. raf- is the initial protein kinase in the mapk signal transduction pathway which phosphorylates subsequent map kinase/extracellular signal-regulated kinase kinase and. we have recently in detail described that activation of mapk-mediated signal transduction is associated with upregulation of et receptors in cerebral vasculature and that et receptor expression is enhanced in ischemic stroke. the importance of mapk signaling in the pathophysiology of ischemic stroke has been widely studied. increased erk1/ phosphorylation has been observed in the ischemic area after both transient and permanent middle cerebral occlusion, as well as after global ischemia. consequently, inhibitors of erk1/ and mek1/ have been effective in reducing the infarct size in cerebral ischemia, and in sah. erk1/ is also activated in the cerebral arteries of the ischemic brain, pointing towards a role in vascular alterations. however, it is not known if the risk factor shs per se may alter et receptor expression in cerebral arteries and if this is associated with intracellular signaling via the raf/erk/mapk pathway. the present study was designed, using an in vivo rat passive smoke exposure model, to demonstrate that cigarette smoke may upregulate cerebrovascular et receptors, and to examine the intracellular signal mechanisms of shs-induced enhanced et receptor expression by in vivo treatment with a specific raf- inhibitor.  general there was no significant difference in cerebral artery contractile responses to k+, sarafotoxin c and et- after or weeks in shs exposed rats as compared to rats exposed to fresh air for a similar time period. therefore, we only present detailed results from the weeks of exposure to shs. the cerebral arteries were from groups of fresh air and shs for and weeks. k+-induced responses were expressed as absolute values of contraction. s6c and et-1-induced responses were expressed as percentage of k+-induced contraction. emax, maximal contraction; shs, secondhand smoke exposure. effects of shs on et receptor-mediated contractions in cerebral artery the contraction elicited by k+ was used as a reference for the contractile capacity. k+- induced contractile responses did not differ significantly in artery segments from fresh air, shs and shs plus inhibitor groups. the etb receptor-mediated contraction was examined using the specific etb receptor agonist s6c, which has been characterized in detail before using the etb receptor antagonist irl. the vasoconstriction induced by a combined eta and etb receptor agonist et- was studied after desensitizing the etb receptors with s6c prior to adding et- leaving only eta receptors to respond. this has been verified by use of the selective eta receptor antagonist fr. the cerebral arteries were from groups of fresh air, shs and shs treatment with gw for weeks. k+-induced responses were expressed as absolute values of contraction. s6c and et-1-induced responses were expressed as percentage of k+-induced contraction. *p < versus fresh air group. emax, maximal contraction; pec negative logarithm of the concentration that produces 50% of the maximal contractile effect; shs, secondhand smoke exposure; gw, gw. s6c only induced a slight contraction in cerebral arteries of fresh air exposed rats. there was no significant difference in s6c-induced contractions between shs and fresh air groups. after desensitization of etb receptors, cumulative administration of et- induced potent contraction of fresh arteries in a concentration-dependent manner with an emax of ± 9%. following shs the concentration-response curve showed an increased emax with no significant difference in pec values. this indicates that the efficacy of the response is increased after shs exposure. effects of shs on et receptor mrna and protein expressions the mrna and protein levels of etb and eta receptors in cerebral arteries were measured by real-time pcr and western blot, respectively. the standard curves of each primer pair in the qpcr had almost similar slopes, indicating that gapdh and receptor cdnas were amplified with the same efficiency. the values of each slope were close to, meaning that the amplification efficiencies were almost optimal. there was no significant contaminating nucleic acid in blank control samples. the etb receptor mrna expression remained unaltered after shs exposure as compared to control. the protein level of etb receptor relative to β-actin was ± in fresh air exposed rats, and ± in the shs exposed group. these results were in concert with the functional myograph studies. the mrna level for the eta receptor relative to gapdh was significantly elevated after shs in cerebral arteries. the level of eta receptor protein was ± relative to β-actin in the fresh air group and enhanced to ± after shs. taken together, the results demonstrate that shs induces eta receptor upregulation. mapk signal pathway studies to investigate the underlying intracellular signal transduction mechanisms associated with the shs-induced increase in eta receptor expression, we first examined the mrna levels of several key protein kinases such as raf- erk erk p38α and jnk by real-time pcr. the results showed that shs increased mrna levels of raf- erk and erk as compared to the fresh air group. shs had no effect on p38α and jnk mrna levels. in addition, we examined the phosphorylated -raf- p-erk1/ p-p and p-jnk, and their total protein expressions by western blot in rat cerebral vessels. total raf- and erk1/ protein were unaltered in the shs group compared to fresh air group. the phosphorylated protein of raf- was enhanced to ± from ±. the level of p-erk1/ protein relative to erk1/ in fresh air and shs animals were ± and ±, respectively. in contrast, the protein level of p-p was ± and ±; the level of p-jnk protein was ± and ±. we did not find any significant difference in protein expression of p-p and p-jnk between fresh air and shs groups. the results show that shs induces enhanced expression of cerebrovascular eta receptors via the raf/erk1/ activation, but does not appear to involve jnk or p pathways in the present experimental conditions. effect of gw on shs-induced effects in order to further understand the role of raf/erk/mapk signal pathway, we studied the inhibitory effect of daily gw administration on shs-induced responses. the results showed that the raf- and erk mrna levels in cerebral arteries were significantly lower after gw treatment in shs exposed animals. erk mrna showed a somewhat lower level after inhibition which was not different from the shs group. the mrna levels of other protein kinases remained unaltered by gw treatment in shs. western blotting confirmed the mrna results. there was a significant decrease in the p-raf- and p-erk1/ protein level after gw treatment as compared with that of shs. the p-p and p-jnk proteins remained unchanged between the smoke exposure and treatment groups. the protein level of p-p was ± and ±; the level of p-jnk protein was ± and ±, respectively. shs or shs with the raf- inhibitor did not modify the weak s6c-induced contraction after weeks of shs combined with daily administration of gw. in contrast, treatment with gw markedly attenuated shs-induced enhanced cerebral vasoconstriction elicited by et- at eta receptors. the emax of the et- induced concentration-contractile curve in gw treatment group was now the same as that seen in rats exposed to fresh air for weeks. the mrna and protein levels of et receptors were also determined after treatment with raf- inhibitor. the etb receptor mrna and protein levels were unchanged in the inhibitor group as compared to shs or fresh air groups. the mrna level of eta receptors was significantly reduced after inhibition of raf/erk/mapk in the shs group. the protein level of eta receptor was lower in the gw treatment group but did not reach statistical significance. discussion 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 p 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. 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. 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 weeks to show eta receptor changes. two or weeks of shs did not alter et receptor-mediated vasoconstriction in cerebral arteries. after 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 receptors and/or increased sensitivity of cerebral vessels in response to receptor agonist. 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 receptor via 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- levels in blood and csf are increased in stroke; this may be further translated to an enhanced receptor-mediated contraction in cerebral arteries. transcriptional upregulation of eta and etb receptors has been reported in rat cerebral arteries after using some injury models like experimental cerebral ischemia and organ culture. in all cases, the receptor upregulation occurred in the smooth muscle cells. the similar findings were confirmed in cerebral vessels from ischemic stroke patients. 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. particularly for erk1/ 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/ in cerebral arteries after mcao and cerebral ischemia. on this basis, the involvement of erk1/ pathway was assessed in the contractile receptor upregulation in artery culture. recently, a number of mapk inhibitors were used to compare their ability to prevent the upregulation of various cerebrovascular vasoconstrictor receptors during organ culture. in the present study we demonstrated shs exposure induced erk1/ signaling activation by enhanced erk1/ phosphorylation. moreover, we showed that shs upregulated eta receptors in rat cerebral arteries. it means shs-induced eta upregulation occurs through erk1/ activation. meanwhile, we used a raf- inhibitor gw and confirmed that it is raf/erk1/ signaling involved in the shs-induced receptor changes, but not jnk or p pathway. this hypothesis is also supported by our recent in vitro discovery in cerebral arteries exposed to lipid-soluble smoke particles. raf- is associated ubiquitously in the raf/mek/erk pathway. raf phosphorylates mek1/ which in turn phosphorylates and activates erk1/ and then leads to activation of transcription factors. the erk1/ pathway is a major effector of raf. transient activation of raf- 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. we chose the raf- inhibitor gw to further demonstrate the involvement of erk in the et receptor upregulation after shs. the specificity and efficacy of gw for inhibiting raf- in-vivo has been established in previous studies. lakey et al. and chin et al. reported that gw is a potent raf- inhibitor and examined the effect of gw on purified raf- and confirmed that gw selectively inhibits raf- in vivo. in the present study, gw 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- and erk1/ was increased after shs, but the total raf- or erk1/ 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- and erk1/ suggests the raf/erk1/ pathway has been activated. the kinases elicit some of their effects through phosphorylation of transcriptional regulation. currently, raf- inhibitor gw reduced phosphorylation of erk1/ as well as raf- the gw5074-induced declined phosphorylation of erk1/ should be attributed to the upstream inhibition of erk1/ however, the reason to explain the reduced raf- phosphorylation is not sure. we think it may be some upstream influences or feedback mechanisms when blocking raf- activity by gw it could be a partial reason for decreased raf- phosphorylation. moreover, we performed in vivo treatment in the animals. it may also be some possible indirect effects of gw that altered raf- phosphorylation when administrated with the inhibitor in vivo. however, the overall data agree with the involvement of raf/erk/mapk in shs.  the present study is the first to show that passive smoke exposure upregulates eta, but not etb receptors, in rat cerebral arteries. the upregulation of eta receptors occurs via activation of the raf/erk/mapk pathway. this mechanism may provide new options for treatment of shs-associated cerebrovascular diseases. 
compared to fresh air exposed rats, cerebral arteries from shs rats exhibited stronger contractile responses mediated by endothelin type a receptors. the results suggest that shs upregulates eta, but not etb receptors in vivo. the mrna and protein expressions for endothelin receptors in cerebral arteries were studied by real-time pcr and western blot. the expressions of mrna and protein for eta receptors in the cerebral arteries from shs rats were higher than that in control. shs upregulates cerebrovascular eta receptors via the raf/erk/mapk pathway, which provides novel understanding of mechanisms involved in shs-associated stroke. after shs exposure, the mrna levels of raf- and erk1/ the protein expression of phosphorylated -raf- and p-erk1/ were increased. shs did not affect endothelin type b receptor-mediated contractions, mrna or protein levels. raf- inhibitor, gw suppressed the enhanced eta receptor-mediated contraction, mrna and protein levels induced by shs. in addition, gw inhibited the shs-caused increased mrna and phosphorylated protein levels of raf- and erk1/ suggesting that shs induces activation of the raf/erk/mapk pathway. the present study established an in vivo rat secondhand cigarette smoking model and examined the hypothesis that shs upregulates endothelin receptors with increased cerebrovascular contraction via the raf/extracellular signal-regulated kinase /mitogen-activated protein kinases pathway.
 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 model and examined the hypothesis that shs upregulates endothelin receptors with increased cerebrovascular contraction via the raf/extracellular signal-regulated kinase /mitogen-activated protein kinases pathway.  rats were exposed to shs for up to 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 mediated by endothelin type a receptors. the expressions of mrna and protein for eta receptors in the cerebral arteries from shs rats were higher than that in control. shs did not affect endothelin type b 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- and erk1/ the protein expression of phosphorylated -raf- and p-erk1/ were increased. raf- inhibitor, gw suppressed the enhanced eta receptor-mediated contraction, mrna and protein levels induced by shs. in addition, gw inhibited the shs-caused increased mrna and phosphorylated protein levels of raf- and erk1/ suggesting that shs induces activation of the raf/erk/mapk pathway.  shs upregulates cerebrovascular eta receptors via the raf/erk/mapk pathway, which provides novel understanding of mechanisms involved in shs-associated stroke. 