BACKGROUND
in the human cardiovascular system, endothelin- <dig>  is the most important isoform, which induces long-lasting vasoconstriction and stimulates proliferation of vascular smooth muscle cells   <cit> .
et- <dig> acts on two g-protein coupled receptors: endothelin type a  and endothelin type b , and plays an important role in hypertension, vascular remodelling, cardiac hypertrophy and coronary artery disease  <cit> .
the eta receptors locate on vsmcs and mediate vasoconstriction, while the etb receptors primarily locate in vascular endothelial cells and mediate transient vasodilation in vivo  <cit> .
however, a subpopulation of contractile etb receptors exist in the vsmcs and mediate vasoconstriction  <cit> .
the eta receptor activates g proteins of gq/ <dig> and g12/ <dig>  which results in the contractile and proliferation effects in vsmcs through activation of diverse signaling molecules such as phospholipase c , intracellular ca2+, protein kinase c , and extracellular signal-regulated kinase  <dig> and  <dig> .
whereas, the etb receptor stimulates the gi and the gq/ <dig> families in vsmcs and endothelial cells  <cit> .
et- <dig> is non-selective agonist for both eta and etb receptors, which may result in receptor signal cross-talk in vascular physiology and pathology.
however, there is limited knowledge about this.
erk1/ <dig>  also termed p44/ <dig> mapk , is one of the members of mapk superfamily, which includes a family of serine/threonine kinase associated with vsmcs contraction, proliferation, migration, differentiation, adhesion, collagen deposition and survival  <cit> .
activation of either the eta or the etb receptor results in phosphorylation of erk1/ <dig>  which is an important regulator for cellular proliferation, migration, differentiation and vascular smooth muscle constriction  <cit> .
a mapk kinase  is required for the erk1/ <dig> phosphorylation of both threonine and tyrosine residues  <cit> .
in the activated form, erk1/ <dig> transmits extracellular stimuli by phosphorylating a variety of substrates including transcription factors and kinases.
there is a paucity of knowledge on intracellular signal mechanisms that et- <dig> leads to activation of erk1/ <dig> in human vsmcs.
non-receptor tyrosine kinase c-src-independent small g protein ras-raf-dependent mechanisms have been reported to mediate et-1-induced erk1/ <dig> phosphorylation in cultured mouse vsmcs  <cit> .
intracellular ca2+ signals are required for mapk/erk1/ <dig> activation induced by angiotensin ii in vsmcs  <cit> .
however, et-1-induced vasoconstriction is not affected by calcium channel blockers  <cit> .
thus, ca2+-independent contraction is suggested to be associated with pkc, phosphoinositide 3-kinase , rho kinase and mapk  <cit> .
the present study was designed, by using a series of specific pharmacological inhibitors, to explore the intracellular signal mechanisms that et- <dig> leads to activation of erk1/ <dig> in human vsmcs with special focus on the receptor signalling.
we have demonstrated that eta receptors predominate over etb receptors in mediating et-1-induced activation of erk1/ <dig> in human vsmcs.
this activation is associated with pkc, pka and pi3k activities, but not intracellular ca2+ signalling.
RESULTS
time course and concentration-dependent activation of erk1/ <dig> induced by et-1
et-1-induced activation of erk1/ <dig> was examined in human aortic smooth muscle cells  at different time points and et- <dig> concentrations.
there was a  <dig>  fold  increase of phosphorylated erk1/ <dig> in cells exposed to  <dig> μm of et- <dig> for  <dig> min; the enhancement reached a peak  at  <dig> min after exposure to et- <dig> .
thereafter, the activities of erk1/ <dig> induced by et- <dig> rapidly declined, and returned to baseline control value at  <dig> min after stimulation.
as verified by western blot , there was an increase in perk1/ <dig> after et- <dig> treatment.
the concentration effects of et- <dig> on erk1/ <dig> activation were investigated at  <dig> min.
it showed that et- <dig> induced activation of erk1/ <dig> in a concentration-dependent manner from  <dig> nm to  <dig> μm .
roles of endothelin receptors in mediating et-1-induced activation of erk1/2
the roles of eta and etb receptors in mediating et-1-induced activation of erk1/ <dig> were studied by using bosentan , bq <dig> , and bq <dig> .
to clarify if the etb receptors in hasmcs were involved in et-1- induced activation of erk1/ <dig>  sarafotoxin 6c , a selective etb receptor agonist was employed and the phosphorylation of erk1/ <dig> was examined by immunofluorescence and western blot .
in figure 2b, there was a slight elevation of phosphorylated erk1/ <dig>  as observed at  <dig> min after exposure to  <dig> μm of s6c.
this peaked at  <dig> min , and quickly declined at  <dig> min .
this slight transient increase of phosphorylated erk1/ <dig> was also produced by  <dig> nm of s6c and verified by western blot for perk1/ <dig> .
bq <dig> and bosentan significantly inhibited the increase in perk1/ <dig> activities, while the etb receptor antagonist bq <dig> had no significant effect .
the increase in phosphorylated erk1/ <dig> was significantly inhibited by  <dig> μm of bq <dig> , which is consistent with the results of phosphoelisa assay  and western blot  .
et-1-induced erk1/ <dig> activation was also significantly inhibited by combination of bq <dig> and bq <dig> by  <dig> % , by  <dig> %  and by  <dig> % .
compared to bq <dig>  a further inhibitory effect was seen in combination of bq <dig> and bq <dig> .
bosentan at  <dig> μm and  <dig> μm significantly inhibited et-1- induced activation of erk1/ <dig> by  <dig> % and  <dig> %, respectively .
at  <dig> μm bosentan had a stronger inhibitory effect on et-1-induced activation of erk1/ <dig> than either bq <dig> or combination of bq <dig> and bq <dig> .
this indicated that etb receptor antagonist bq <dig> had no significant inhibitory effect on et-1-induced activation of erk1/ <dig> in the absence of eta receptor antagonist bq <dig>  while bosentan, a dual et receptor agonist or combined use of bq <dig> and bq <dig>  further decreased et-1-induced activation of erk1/ <dig>
role of the mek on et-1-induced activation of erk1/2
three different mek/erk kinase inhibitors were used to study et-1-induced activation of erk1/ <dig> in hasmcs.
as shown in figure 3a and 3b, u <dig>  a potent mek1/ <dig> inhibitor, at the concentration  <dig> and  <dig> μm completely inhibited et-1-induced phosphorylation of erk1/ <dig> from 258% to 87% and 63%, respectively.
sl <dig>  another selective inhibitor of mek <dig> and mek <dig> had similar degree of inhibitory effects .
pd <dig>  a selective inhibitor of mek <dig>  only partially inhibited et-1-induced phosphorylation of erk1/ <dig> from 258% to 153% at  <dig> μm, and to 145% at  <dig> μm, respectively .
this suggests that both mek <dig> and mek <dig> are required for et- <dig> to activate erk1/ <dig> in hasmcs.
this is further supported by phosphoelisa assay  and western blot .
compared to pd <dig>  u <dig> at  <dig> μm had a significant stronger inhibitory effect .
to clarify whether u <dig> also inhibits phosphorylation of erk1/ <dig> in untreated control cells, the phosphoelisa assay was used.
it showed that in untreated control hasmcs, u <dig> at  <dig> μm did not significantly modify erk1/ <dig> activity .
in et-1-treated hasmcs, u <dig> significantly decreased the phosphorylated erk1/ <dig> level at the same concentration .
roles of pkc/pka and small g proteins on et-1-induced activation of erk1/2
to further determine the upstream signaling involved in the mek/erk pathway, we used pharmacological inhibitors and examined the effects of pkc inhibitors , pkc-delta inhibitor , pka specific inhibitor , and pi3k inhibitor  on et-1-induced perk1/ <dig> activities .
the activation of erk1/ <dig> was significantly inhibited by  <dig> nm of staurosporin ,  <dig> μm of gf 109203x ,  <dig> μm of rottlerin ,  <dig> μm of h- <dig> , and  <dig> μm of wortmannin , respectively .
similar, results were obtained in the phosphoelisa assay .
role of extracellular ca2+ influx or intracellular ca2+ release in mediating et-1-induced activation of erk1/ <dig> in hasmcs
ca2+, a second messenger, has a central role in activation of various key cellular responses, including muscle contraction, cell proliferation, migration and adhesion  <cit> .
to evaluate the role of intracellular ca2+signaling in mediating et-1-induced activation of erk1/ <dig>  nifedipine was used to block external ca2+ influx through l-type ca2+ channels,  <dig> mm of egta was employed to chelate extracellular ca2+, and  <dig> μm of thapsigargin was used to cause intracellular ca2+ stores to become depleted.
kn- <dig>  a calcium-calmodulin dependent protein kinase ii  inhibitor was also examined .
the activation of erk1/ <dig> was not affected by l-type ca2+ channel blocker , chelating extracellular ca2+ , abolishing intracellular ca2+ release , or inhibition of camkii .
replacing the medium with calcium-free pbs  did not inhibit et-1-induced activation of erk1/ <dig>  these indicated that extracellular ca2+ influx and ca2+ released from internal stores were not necessarily required for the et-1-induced phosphorylation of erk1/ <dig> in hasmcs.
this is further supported by the results from phosphoelisa assay .
to identify whether extracellular ca2+ was chelated or ca2+ influx was decreased in our experiments, we used  <dig> μm of thapsigargin to induce extracellular ca2+ influx through store-operated ca2+ channels   <cit> .
we found that thapsigargin resulted in an activation of erk1/ <dig> in hasmcs as reported in rbl- <dig> cells  <cit> .
the activation of erk1/ <dig> was abolished by  <dig> mm of egta .
this suggests that  <dig> mm of egta can effectively chelate extracellular ca2+and decrease ca2+ influx in our experiments.
discussion
the present study has revealed that et- <dig> acts primarily via the eta receptors to induce phosphorylation of erk1/ <dig> in hasmcs.
the et-1-induced response requires intracellular signal molecule pkc, pka and pi3k activities, while it is independent of intracellular calcium signaling.
et-1-induced activation of erk1/ <dig> in hasmcs
erk1/ <dig> are important regulators of cell proliferation and migration in vsmcs  <cit> .
these basic cellular functions are important for the formation of the neointima in pathologic states such as atherosclerosis.
many stimuli such as mechanical stretch, growth factors, cytokines and activation of g protein-coupled receptors, can result in phosphorylation of erk1/ <dig> and its signal pathways.
recent studies have demonstrated that erk1/ <dig> mapk pathways regulate ca2+-dependent and ca2+-independent contraction of vsmcs  <cit> .
intracellular erk1/ <dig> mapk signal mechanisms play important roles in vascular pathology and in the development of cardiovascular disease  <cit> .
et- <dig> not only remains the most potent and long-lasting vasoconstrictor of human vessels, it also induces proliferation of vascular smooth muscle cells through activation of erk1/ <dig>  <cit>  in pulmonary hypertension, atherosclerosis, heart failure and restenosis  <cit> .
in human arterial smooth muscle cells, et-1-induced activation of erk1/ <dig> is much weaker in aortic artery than in coronary artery  <cit> .
this implies that small arteries are more sensitive than large arteries.
unlike angiotensin ii, which shows a rapid and transient increase in activities of erk1/ <dig>  <cit> , et- <dig> induced a long-lasting phosphorylation of erk1/ <dig> with a peaked at  <dig> min and declined to baseline after  <dig> min in present study.
the activation of erk1/ <dig> by et- <dig> might contribute to vsmc proliferation in formation of new intima and thus it may contribute to serve as an early "switch-on" mechanism for cardiovascular disease development  <cit> .
roles of et receptors in activation of erk1/ <dig> in hasmcs
the physiological and pathological effects of et- <dig> are mediated through two g protein-coupled receptors, eta and etb.
in human vasculature, eta receptors predominate on the smooth muscle cells and mediate constriction, whereas etb receptors are expressed less than 15% on these cells  <cit> .
in-vivo studies suggest that both subtypes of endothelin receptors can mediate vasoconstriction in human resistance and capacitance vessels  <cit> .
in the present study, we found that eta predominately mediated et-1-induced activation of erk1/ <dig>  although some activation of erk1/ <dig> was obtained with the etb-selective agonist, s6c, the maximum response produced to s6c was transient and less than 20% of the et- <dig> effect.
in addition, bq <dig>  a selective antagonist of the eta receptor  <cit> , but not etb receptor antagonist bq <dig>  significantly inhibited the activation of erk1/ <dig> induced by et- <dig>  suggesting that et-1-induced activation of erk1/ <dig> is predominately mediated by etareceptors.
compared to bq <dig>  a further inhibition of et-1-induced activation of erk1/ <dig> was obtained in combination of bq <dig> and bq <dig>  bosentan, a dual eta and etb receptor antagonist had a significant stronger inhibitory effect on et-1-induced activation of erk1/ <dig> than either bq <dig> or the combination of bq <dig> and bq <dig>  these results suggest that et receptor dimerization  <cit>  might also occur in human vsmcs in the presence of et- <dig> as a bivalent ligand connecting two receptors  <cit>  and that the receptor cross-talk is involved in the et- <dig> effect.
however, this requires more studies to verify.
upstream intracellular signal molecules involved in et-1-induced activation of erk1/2
erk1/ <dig> activation requires a sequential activation of ras, raf and mek signal cascades  <cit> .
mek inhibitors  were used to investigate the role of upstream mek in et-1-induced activation of erk1/ <dig>  u <dig>  a highly selective inhibitor of mek1/ <dig> had the same potency as sl <dig> , and completely inhibited et-1-induced activation of erk1/ <dig>  whereas, pd <dig>  a selective mek <dig> inhibitor, only partially inhibited et-1-induced activation of erk1/ <dig>  pkc, a family of serine/threonine kinases, may be involved in the intracellular signal transduction of mek/erk1/ <dig> induced by et- <dig>  pka is an important second messenger.
cyclic amp-independent activation of pka by et- <dig> has been observed in rat aortic smooth muscle cells  <cit> .
on the other hand, g-protein-coupled receptor signaling can be mediated through various small g proteins.
the ras/raf pathway is found to be a proximal regulator of mek  <cit> .
pi3k, another downstream effector of ras  <cit> , has been linked to a diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival  <cit> .
by using selective inhibitors, the present study revealed that pkc, pka and pi3k were involved in activation of erk1/ <dig> induced by et- <dig> in hasmcs, which may provide targets for drug discovery  <cit> .
intracellular ca2+ signaling was not required for et-1-induced activation of erk1/2
et- <dig> stimulates phospholipase c-dependent hydrolysis of pip <dig>  through g-protein coupled receptors, leading to the generation of inositol  <dig> ,5-trisphosphate  and diacylglycerol , which are involved in intracellular ca2+ mobilization and pkc activation  <cit> .
recently, growing evidence has shown that ca2+ signaling is critical for activation of erk1/ <dig> induced by angiotensin ii in vsmcs  <cit> .
however, the role of intracellular ca2+ signaling in et-1-induced activation of erk1/ <dig> in human vsmcs remains unclear.
it has been reported that the activation of l-type ca2+ channels contributes to et-1-induced sustained phase of the ca2+ response and the ability to generate force  <cit> .
unlike angiotensin ii, the present study revealed that extracellular ca2+ influx through l-type ca2+ channels did not participate in et-1-induced activation of erk1/ <dig> in human vsmcs.
to further investigate the involvement of intracellular ca2+ through other ca2+ channels, which are suggested to be involved in et-1-mediated contractions of vsmc  <cit>  and mitogenesis  <cit> ,  <dig> mm of egta was used.
extracellular ca2+ chelation by egta did not affect activation of erk1/ <dig> induced by et- <dig>  et-1-induced ca2+ release from intracellular stores is triggered by the binding of ip <dig> to receptors on the sarcoplasmic reticulum .
depletion of intracellular ca2+ stores can lead to a local ca2+ flux through store-operated ca2+ channels , which has been reported to initiate the activation of erk1/ <dig> in rbl- <dig> cells  <cit> .
therefore, in our studies, thapsigargin, an inhibitor to the sr ca2+-atpase pump, which results in ca2+ release and depletion from internal stores, was applied together with  <dig> mm of egta.
the results showed that erk1/ <dig> activation by et- <dig> did not require the participation of intracellular ca2+ release.
studies have indicated that the camkii pathway mediates g-protein coupled receptor ligand-depedent activation of erk1/ <dig> in cultured vsm cells  <cit> .
however, we observed that camkii pathway was probably not involved in the et-1- induced activation of erk1/ <dig> in human vsmcs as based on kn- <dig> inhibition experiment.
using receptor-operated ca2+ channel blockers loe  <dig> and sk&f  <dig>  and l-type ca2+ channels blocker nifedipine, kawanabe et al noted that et-1-induced erk1/ <dig> activiation involved a ca2+ influx-dependent cascade through ca2+permeable nonselective cation channels  and socc, and a ca2+influx-independent cascade in rabbit carotid artery vsmcs  <cit> .
the studies showed that maximal effective concentration of nifedipine has only 10% of the inhibition on et-1-induced increases in erk1/ <dig> activity.
however, we did not find significant changes of phosphorylated erk1/ <dig> induced by et- <dig> after treatment with nifedipine or chelation of extracellular ca2+.
CONCLUSIONS
in conclusion, we have demontrated that et-1-induced activation of erk1/ <dig> in human vsmcs is predominantly mediated by eta receptors through upstream signal molecule pkc, pka and pi3k, while it is independent of camkii and intracellular ca2+ signaling.
the endothelin system plays key roles in hypertension, stoke and myocardial infarction.
understanding the intracellular signaling mechanisms of endothelin receptors may provide new strategies for developing new drugs for cardiovascular diseases.

 in the human cardiovascular system, endothelin- is the most important isoform, which induces long-lasting vasoconstriction and stimulates proliferation of vascular smooth muscle cells. et- acts on two g-protein coupled receptors: endothelin type a and endothelin type b, and plays an important role in hypertension, vascular remodelling, cardiac hypertrophy and coronary artery disease. the eta receptors locate on vsmcs and mediate vasoconstriction, while the etb receptors primarily locate in vascular endothelial cells and mediate transient vasodilation in vivo. however, a subpopulation of contractile etb receptors exist in the vsmcs and mediate vasoconstriction. the eta receptor activates g proteins of gq/ and g12/ which results in the contractile and proliferation effects in vsmcs through activation of diverse signaling molecules such as phospholipase c, intracellular ca2+, protein kinase c, and extracellular signal-regulated kinase and. whereas, the etb receptor stimulates the gi and the gq/ families in vsmcs and endothelial cells. et- is non-selective agonist for both eta and etb receptors, which may result in receptor signal cross-talk in vascular physiology and pathology. however, there is limited knowledge about this. erk1/ also termed p44/ mapk, is one of the members of mapk superfamily, which includes a family of serine/threonine kinase associated with vsmcs contraction, proliferation, migration, differentiation, adhesion, collagen deposition and survival. activation of either the eta or the etb receptor results in phosphorylation of erk1/ which is an important regulator for cellular proliferation, migration, differentiation and vascular smooth muscle constriction. a mapk kinase is required for the erk1/ phosphorylation of both threonine and tyrosine residues. in the activated form, erk1/ transmits extracellular stimuli by phosphorylating a variety of substrates including transcription factors and kinases. there is a paucity of knowledge on intracellular signal mechanisms that et- leads to activation of erk1/ in human vsmcs. non-receptor tyrosine kinase c-src-independent small g protein ras-raf-dependent mechanisms have been reported to mediate et-1-induced erk1/ phosphorylation in cultured mouse vsmcs. intracellular ca2+ signals are required for mapk/erk1/ activation induced by angiotensin ii in vsmcs. however, et-1-induced vasoconstriction is not affected by calcium channel blockers. thus, ca2+-independent contraction is suggested to be associated with pkc, phosphoinositide 3-kinase, rho kinase and mapk. the present study was designed, by using a series of specific pharmacological inhibitors, to explore the intracellular signal mechanisms that et- leads to activation of erk1/ in human vsmcs with special focus on the receptor signalling. we have demonstrated that eta receptors predominate over etb receptors in mediating et-1-induced activation of erk1/ in human vsmcs. this activation is associated with pkc, pka and pi3k activities, but not intracellular ca2+ signalling.  time course and concentration-dependent activation of erk1/ induced by et-1 et-1-induced activation of erk1/ was examined in human aortic smooth muscle cells at different time points and et- concentrations. there was a fold increase of phosphorylated erk1/ in cells exposed to μm of et- for min; the enhancement reached a peak at min after exposure to et-. thereafter, the activities of erk1/ induced by et- rapidly declined, and returned to baseline control value at min after stimulation. as verified by western blot, there was an increase in perk1/ after et- treatment. the concentration effects of et- on erk1/ activation were investigated at min. it showed that et- induced activation of erk1/ in a concentration-dependent manner from nm to μm. roles of endothelin receptors in mediating et-1-induced activation of erk1/2 the roles of eta and etb receptors in mediating et-1-induced activation of erk1/ were studied by using bosentan, bq, and bq. to clarify if the etb receptors in hasmcs were involved in et-1- induced activation of erk1/ sarafotoxin 6c, a selective etb receptor agonist was employed and the phosphorylation of erk1/ was examined by immunofluorescence and western blot. in figure 2b, there was a slight elevation of phosphorylated erk1/ as observed at min after exposure to μm of s6c. this peaked at min, and quickly declined at min. this slight transient increase of phosphorylated erk1/ was also produced by nm of s6c and verified by western blot for perk1/. bq and bosentan significantly inhibited the increase in perk1/ activities, while the etb receptor antagonist bq had no significant effect. the increase in phosphorylated erk1/ was significantly inhibited by μm of bq, which is consistent with the results of phosphoelisa assay and western blot. et-1-induced erk1/ activation was also significantly inhibited by combination of bq and bq by %, by % and by %. compared to bq a further inhibitory effect was seen in combination of bq and bq. bosentan at μm and μm significantly inhibited et-1- induced activation of erk1/ by % and %, respectively. at μm bosentan had a stronger inhibitory effect on et-1-induced activation of erk1/ than either bq or combination of bq and bq. this indicated that etb receptor antagonist bq had no significant inhibitory effect on et-1-induced activation of erk1/ in the absence of eta receptor antagonist bq while bosentan, a dual et receptor agonist or combined use of bq and bq further decreased et-1-induced activation of erk1/ role of the mek on et-1-induced activation of erk1/2 three different mek/erk kinase inhibitors were used to study et-1-induced activation of erk1/ in hasmcs. as shown in figure 3a and 3b, u a potent mek1/ inhibitor, at the concentration and μm completely inhibited et-1-induced phosphorylation of erk1/ from 258% to 87% and 63%, respectively. sl another selective inhibitor of mek and mek had similar degree of inhibitory effects. pd a selective inhibitor of mek only partially inhibited et-1-induced phosphorylation of erk1/ from 258% to 153% at μm, and to 145% at μm, respectively. this suggests that both mek and mek are required for et- to activate erk1/ in hasmcs. this is further supported by phosphoelisa assay and western blot. compared to pd u at μm had a significant stronger inhibitory effect. to clarify whether u also inhibits phosphorylation of erk1/ in untreated control cells, the phosphoelisa assay was used. it showed that in untreated control hasmcs, u at μm did not significantly modify erk1/ activity. in et-1-treated hasmcs, u significantly decreased the phosphorylated erk1/ level at the same concentration. roles of pkc/pka and small g proteins on et-1-induced activation of erk1/2 to further determine the upstream signaling involved in the mek/erk pathway, we used pharmacological inhibitors and examined the effects of pkc inhibitors, pkc-delta inhibitor, pka specific inhibitor, and pi3k inhibitor on et-1-induced perk1/ activities. the activation of erk1/ was significantly inhibited by nm of staurosporin, μm of gf 109203x, μm of rottlerin, μm of h-, and μm of wortmannin, respectively. similar, results were obtained in the phosphoelisa assay. role of extracellular ca2+ influx or intracellular ca2+ release in mediating et-1-induced activation of erk1/ in hasmcs ca2+, a second messenger, has a central role in activation of various key cellular responses, including muscle contraction, cell proliferation, migration and adhesion. to evaluate the role of intracellular ca2+signaling in mediating et-1-induced activation of erk1/ nifedipine was used to block external ca2+ influx through l-type ca2+ channels, mm of egta was employed to chelate extracellular ca2+, and μm of thapsigargin was used to cause intracellular ca2+ stores to become depleted. kn- a calcium-calmodulin dependent protein kinase ii inhibitor was also examined. the activation of erk1/ was not affected by l-type ca2+ channel blocker, chelating extracellular ca2+, abolishing intracellular ca2+ release, or inhibition of camkii. replacing the medium with calcium-free pbs did not inhibit et-1-induced activation of erk1/ these indicated that extracellular ca2+ influx and ca2+ released from internal stores were not necessarily required for the et-1-induced phosphorylation of erk1/ in hasmcs. this is further supported by the results from phosphoelisa assay. to identify whether extracellular ca2+ was chelated or ca2+ influx was decreased in our experiments, we used μm of thapsigargin to induce extracellular ca2+ influx through store-operated ca2+ channels. we found that thapsigargin resulted in an activation of erk1/ in hasmcs as reported in rbl- cells. the activation of erk1/ was abolished by mm of egta. this suggests that mm of egta can effectively chelate extracellular ca2+and decrease ca2+ influx in our experiments. discussion the present study has revealed that et- acts primarily via the eta receptors to induce phosphorylation of erk1/ in hasmcs. the et-1-induced response requires intracellular signal molecule pkc, pka and pi3k activities, while it is independent of intracellular calcium signaling. et-1-induced activation of erk1/ in hasmcs erk1/ are important regulators of cell proliferation and migration in vsmcs. these basic cellular functions are important for the formation of the neointima in pathologic states such as atherosclerosis. many stimuli such as mechanical stretch, growth factors, cytokines and activation of g protein-coupled receptors, can result in phosphorylation of erk1/ and its signal pathways. recent studies have demonstrated that erk1/ mapk pathways regulate ca2+-dependent and ca2+-independent contraction of vsmcs. intracellular erk1/ mapk signal mechanisms play important roles in vascular pathology and in the development of cardiovascular disease. et- not only remains the most potent and long-lasting vasoconstrictor of human vessels, it also induces proliferation of vascular smooth muscle cells through activation of erk1/ in pulmonary hypertension, atherosclerosis, heart failure and restenosis. in human arterial smooth muscle cells, et-1-induced activation of erk1/ is much weaker in aortic artery than in coronary artery. this implies that small arteries are more sensitive than large arteries. unlike angiotensin ii, which shows a rapid and transient increase in activities of erk1/, et- induced a long-lasting phosphorylation of erk1/ with a peaked at min and declined to baseline after min in present study. the activation of erk1/ by et- might contribute to vsmc proliferation in formation of new intima and thus it may contribute to serve as an early "switch-on" mechanism for cardiovascular disease development. roles of et receptors in activation of erk1/ in hasmcs the physiological and pathological effects of et- are mediated through two g protein-coupled receptors, eta and etb. in human vasculature, eta receptors predominate on the smooth muscle cells and mediate constriction, whereas etb receptors are expressed less than 15% on these cells. in-vivo studies suggest that both subtypes of endothelin receptors can mediate vasoconstriction in human resistance and capacitance vessels. in the present study, we found that eta predominately mediated et-1-induced activation of erk1/ although some activation of erk1/ was obtained with the etb-selective agonist, s6c, the maximum response produced to s6c was transient and less than 20% of the et- effect. in addition, bq a selective antagonist of the eta receptor, but not etb receptor antagonist bq significantly inhibited the activation of erk1/ induced by et- suggesting that et-1-induced activation of erk1/ is predominately mediated by etareceptors. compared to bq a further inhibition of et-1-induced activation of erk1/ was obtained in combination of bq and bq bosentan, a dual eta and etb receptor antagonist had a significant stronger inhibitory effect on et-1-induced activation of erk1/ than either bq or the combination of bq and bq these results suggest that et receptor dimerization might also occur in human vsmcs in the presence of et- as a bivalent ligand connecting two receptors and that the receptor cross-talk is involved in the et- effect. however, this requires more studies to verify. upstream intracellular signal molecules involved in et-1-induced activation of erk1/2 erk1/ activation requires a sequential activation of ras, raf and mek signal cascades. mek inhibitors were used to investigate the role of upstream mek in et-1-induced activation of erk1/ u a highly selective inhibitor of mek1/ had the same potency as sl, and completely inhibited et-1-induced activation of erk1/ whereas, pd a selective mek inhibitor, only partially inhibited et-1-induced activation of erk1/ pkc, a family of serine/threonine kinases, may be involved in the intracellular signal transduction of mek/erk1/ induced by et- pka is an important second messenger. cyclic amp-independent activation of pka by et- has been observed in rat aortic smooth muscle cells. on the other hand, g-protein-coupled receptor signaling can be mediated through various small g proteins. the ras/raf pathway is found to be a proximal regulator of mek. pi3k, another downstream effector of ras, has been linked to a diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival. by using selective inhibitors, the present study revealed that pkc, pka and pi3k were involved in activation of erk1/ induced by et- in hasmcs, which may provide targets for drug discovery. intracellular ca2+ signaling was not required for et-1-induced activation of erk1/2 et- stimulates phospholipase c-dependent hydrolysis of pip through g-protein coupled receptors, leading to the generation of inositol,5-trisphosphate and diacylglycerol, which are involved in intracellular ca2+ mobilization and pkc activation. recently, growing evidence has shown that ca2+ signaling is critical for activation of erk1/ induced by angiotensin ii in vsmcs. however, the role of intracellular ca2+ signaling in et-1-induced activation of erk1/ in human vsmcs remains unclear. it has been reported that the activation of l-type ca2+ channels contributes to et-1-induced sustained phase of the ca2+ response and the ability to generate force. unlike angiotensin ii, the present study revealed that extracellular ca2+ influx through l-type ca2+ channels did not participate in et-1-induced activation of erk1/ in human vsmcs. to further investigate the involvement of intracellular ca2+ through other ca2+ channels, which are suggested to be involved in et-1-mediated contractions of vsmc and mitogenesis, mm of egta was used. extracellular ca2+ chelation by egta did not affect activation of erk1/ induced by et- et-1-induced ca2+ release from intracellular stores is triggered by the binding of ip to receptors on the sarcoplasmic reticulum. depletion of intracellular ca2+ stores can lead to a local ca2+ flux through store-operated ca2+ channels, which has been reported to initiate the activation of erk1/ in rbl- cells. therefore, in our studies, thapsigargin, an inhibitor to the sr ca2+-atpase pump, which results in ca2+ release and depletion from internal stores, was applied together with mm of egta. the results showed that erk1/ activation by et- did not require the participation of intracellular ca2+ release. studies have indicated that the camkii pathway mediates g-protein coupled receptor ligand-depedent activation of erk1/ in cultured vsm cells. however, we observed that camkii pathway was probably not involved in the et-1- induced activation of erk1/ in human vsmcs as based on kn- inhibition experiment. using receptor-operated ca2+ channel blockers loe and sk&f and l-type ca2+ channels blocker nifedipine, kawanabe et al noted that et-1-induced erk1/ activiation involved a ca2+ influx-dependent cascade through ca2+permeable nonselective cation channels and socc, and a ca2+influx-independent cascade in rabbit carotid artery vsmcs. the studies showed that maximal effective concentration of nifedipine has only 10% of the inhibition on et-1-induced increases in erk1/ activity. however, we did not find significant changes of phosphorylated erk1/ induced by et- after treatment with nifedipine or chelation of extracellular ca2+.  in conclusion, we have demontrated that et-1-induced activation of erk1/ in human vsmcs is predominantly mediated by eta receptors through upstream signal molecule pkc, pka and pi3k, while it is independent of camkii and intracellular ca2+ signaling. the endothelin system plays key roles in hypertension, stoke and myocardial infarction. understanding the intracellular signaling mechanisms of endothelin receptors may provide new strategies for developing new drugs for cardiovascular diseases. 
the activation of erk1/ was examined by immunofluorescence, western blot and phosphoelisa using specific antibody against phosphorylated erk1/ protein. the et-1-induced activation of erk1/ was completely abolished by mek1/ inhibitors u and sl and partially inhibited by the mek inhibitor pd a dual endothelin receptor antagonist bosentan or the eta antagonist bq blocked the et- effect, while the etb antagonist bq had no significant effect. the eta receptors predominate in the et-1-induced activation of erk1/ in human vsmcs, which associates with increments in intracellular pkc, pka and pi3k activities, but not ca2+ signalling. however, a selective etb receptor agonist, sarafotoxin 6c caused a time-dependent erk1/ activation with a maximal effect by less than 20% of the et-1-induced activation of erk1/ increase in bosentan concentration up to μm further inhibited et-1-induced activation of erk1/ and had a stronger inhibitory effect than bq or the combined use of bq and bq to further explore et- intracellular signaling, pkc inhibitors, pkc-delta inhibitor, pka inhibitor, and phosphatidylinositol 3-kinase inhibitor were applied. the inhibitors showed significant inhibitory effects on et-1-induced activation of erk1/ however, blockage of l-type ca2+ channels or calcium/calmodulin-dependent protein kinase ii, chelating extracellular ca2+ or emptying internal ca2+ stores, did not affect et-1-induced activation of erk1/ endothelin- is a potent vasoactive peptide, which induces vasoconstriction and proliferation in vascular smooth muscle cells through activation of endothelin type a and type b receptors. this study was designed to investigate the eta and etb receptor intracellular signaling in human vsmcs and used phosphorylation of erk1/ as a functional signal molecule for endothelin receptor activity. subconfluent human vsmcs were stimulated by et- at different concentrations.
 endothelin- is a potent vasoactive peptide, which induces vasoconstriction and proliferation in vascular smooth muscle cells through activation of endothelin type a and type b receptors. the extracellular signal-regulated kinase and mitogen-activated protein kinases are involved in et-1-induced vsmc contraction and proliferation. this study was designed to investigate the eta and etb receptor intracellular signaling in human vsmcs and used phosphorylation of erk1/ as a functional signal molecule for endothelin receptor activity.  subconfluent human vsmcs were stimulated by et- at different concentrations. the activation of erk1/ was examined by immunofluorescence, western blot and phosphoelisa using specific antibody against phosphorylated erk1/ protein. et- induced a concentration- and time- dependent activation of erk1/ with a maximal effect at min. it declined to baseline level at min. the et-1-induced activation of erk1/ was completely abolished by mek1/ inhibitors u and sl and partially inhibited by the mek inhibitor pd a dual endothelin receptor antagonist bosentan or the eta antagonist bq blocked the et- effect, while the etb antagonist bq had no significant effect. however, a selective etb receptor agonist, sarafotoxin 6c caused a time-dependent erk1/ activation with a maximal effect by less than 20% of the et-1-induced activation of erk1/ increase in bosentan concentration up to μm further inhibited et-1-induced activation of erk1/ and had a stronger inhibitory effect than bq or the combined use of bq and bq to further explore et- intracellular signaling, pkc inhibitors, pkc-delta inhibitor, pka inhibitor, and phosphatidylinositol 3-kinase inhibitor were applied. the inhibitors showed significant inhibitory effects on et-1-induced activation of erk1/ however, blockage of l-type ca2+ channels or calcium/calmodulin-dependent protein kinase ii, chelating extracellular ca2+ or emptying internal ca2+ stores, did not affect et-1-induced activation of erk1/  the eta receptors predominate in the et-1-induced activation of erk1/ in human vsmcs, which associates with increments in intracellular pkc, pka and pi3k activities, but not ca2+ signalling. 