increasing cytosolic calcium ion ( ca ) concentration is essential for the contraction of smooth muscle cells .
the increase results from the influx of ca through the plasma membrane and release of ca from intracellular stores , mainly the sarcoplasmic reticulum ( sr ) [ 14 ] .
phenylephrine ( pe ) or angiotensin ii ( ang ii ) induces receptor - coupled g protein - induced phosphoinositide 3-kinase ( pi3k ) and phospholipase c ( plc ) activation , resulting in ca - dependent vasoconstriction in smooth muscle cells [ 510 ] .
pi3k activity facilitates the production of 3-phosphorylated phosphoinositides such as phosphatidylinositol 3-phosphate ( pi(3)p ) , phosphatidylinositol ( 3,4)-bisphosphate ( pi(3,4)p2 ) , and phosphatidylinositol ( 3,4,5)-triphosphate ( pi(3,4,5)p3 ) [ 11 , 12 ] . among these
, pi(3,4,5)p3 stimulates the l - type ca channel ( 12 ) , a voltage - dependent ca channel that plays an important role in the regulation of vascular tone [ 5 , 12 , 13 ] .
activated plc is an effector in the stimulation of ca release from the endoplasmic reticulum ( er ) or the sr [ 1416 ] .
plc hydrolyzes phosphatidylinositol 4,5-bisphosphate ( pip2 ) into diacylglycerol ( dag ) and inositol ( 3 , 4 , 5)-triphosphate ( ip3 ) .
the latter is released as a soluble structure into the cytosol , where it binds to ip3 receptors in the sr [ 15 , 17 ] .
this binding process increases the cytosolic ca concentration and smooth muscle constriction [ 15 , 16 ] .  
the herb cinnamomi ramulus ( cr ) has traditionally been used in asia and europe to treat maladies involving blood circulation and inflammation . in one study ,
an aqueous extract of cr ameliorated sucrose - induced blood pressure elevation in spontaneously hypertensive rats .
recently , we reported that cr ethanol extract ( cre ) reduces vascular contraction through the inhibition of voltage - dependent ca channels .
the present study explored the suggestion that the vasodilatory effect of cre is related to ca - dependent mechanisms in rat aorta .
all animals were provided with food and water ad libitum and allowed to adapt to the experimental conditions ( temperature , 21  2c ; humidity , 5060% ) for 1 week .
rabbit polyclonal antibodies against phosphorylated plc ( pplc ) , -actin , and anti - rabbit secondary antibody were purchased from santa cruz biotechnology ( santa cruz , calif , usa ) .
pi3k / p85 antibody was purchased from cell signaling technology ( beverly , mass , usa ) .
pe , ang ii , verapamil , nifedipine , fpl64176 , 2 , 4 , 6-trimethyl - n-(meta-3-trifluoromethyl - phenyl)-benzenesulfonamide ( m-3m3fbs ) , u73122 and caffeine were purchased from sigma - aldrich ( st . louis , mo , usa ) .
cr ( twigs of cinnamomum cassia blume ) collected in china in november 2009 was purchased from humanherb ( gyeongsan , korea ) .
a voucher specimen ( cre08 ) has been deposited in the college of oriental medicine , dongguk university . dried cr ( 100  g )
was extracted with 500  ml of 70% ethanol by heating at 75c for 3  h. the extract was filtered through whatman filter paper ( whatman international , maidstone , uk ) to remove the insoluble materials .
after filtration , the extracts were concentrated by rotary evaporation using a model vv2000 apparatus ( heidolph , walpersdorfer , germany ) at a temperature of 75c and then dried using a model fd8508s freeze dryer ( ilshin , busan , korea ) .
the ec50 value of 0.1  mg / ml cre was used in all experiments . in a previous research , cinnamaldehyde and coumarin
also , cinnamaldehyde was known as major active compound of cr for vasodilation , antitumor , and antifungal activity .
all procedures were performed according to protocols approved by the institutional animal care and use committee of dongguk university .
briefly , rats were sacrificed and their thoracic aortas were immediately excised and immersed in ice - cold krebs solution ( 115.0  mm nacl , 4.7  mm kcl , 2.5  mm cacl2 , 1.2  mm mgcl2 , 25.0  mm nahco3 , 1.2  mm kh2po4 , and 10.0  mm dextrose ) .
the aortas were cleaned of all adherent connective tissue and cut into 3  mm long ring segments .
endothelium was removed from the internal surface of each segment by gentle rubbing with forceps .
one triangle was anchored to a stationary support and the other was connected to a ft03 isometric force transducer ( grass , quincy , mass , usa ) .
each vessel ring was incubated in a water - jacketed organ bath ( 10  ml ) that was maintained at 37c and aerated with a mixture of 95% o2 and 5% co2 .
each ring was stretched passively by imposing the optimal resting tension of approximately 2.0  g , which was maintained throughout the experiment .
each endothelium - free aortic ring was allowed to equilibrate in the organ bath for at least 50  min before the experiment involving the contractile response to 5  m ang ii , 1  m pe , 10  m fpl64176 , 5  g / ml m-3m3fbs , or 30  mm caffeine .
endothelium - free rings were used because preliminary experiments ( data not shown ) established that cre relaxes vascular constriction in an endothelium - independent manner .
the denudation of endothelium was assessed by treating the rings with 1  m acetylcholine .
isometric tension was recorded using a powerlab/8sp computerized data acquisition system ( adinstruments , castle hill , nsw , australia ) .
the influence of cre on extracellular ca influx was studied in ca - free krebs solution .
after equilibration of the ring in ca - free krebs solution containing 60 mm kcl , cumulative doses of cacl2 were added ( 0.3 , 0.6 , 1 , 1.5 , 2.5 , 5 , and 10  mm , in order ) with preincubation of cre in organ bath .
the cacl2 dose - dependent maximum constriction of the aortic ring with 60  mm kcl in ca - free krebs solution was expressed as 100% . to determine the influence of cre on ca influx through the l - type ca channel
, aortic rings were pretreated with nifedipine or verapamil before pe contraction , and were preincubated with cre before contraction by fpl64176 . to investigate the inhibitory effect of cre on intracellular ca release by pe in ca - free conditions , and by caffeine in normal krebs solution ,
the transient contraction of cre preincubated aortic rings was measured . to further investigate the relationship with the plc pathway ,
aortic rings were constricted with m-3m3fbs , and were preincubated with u73122 prior to contraction by pe .
a previously described protocol   was used for preparation of protein extract with some modifications .
briefly , endothelium - free aortic rings were contracted with 1  m pe or 5  g / ml m-3m3fbs , and then treated with cre for 30  min .
the aortic rings were quick frozen by immersion in acetone containing 10% trichloroacetic acid ( tca ) and 10  mm dithiothreitol ( dtt ) precooled to 80c .
when used , recovered samples were homogenized in buffer containing 320  mm sucrose , 50  mm tris , 1  mm edta , 1% triton x-100 , 1  mm dtt , and the following protease inhibitors : leupeptin ( 10  g / ml ) , trypsin ( 10  g / ml ) , aprotinin ( 2  g / ml ) , or phenylmethylsulphonyl fluoride ( 100  g / ml ) .
the protein samples were electrophoresed and the resolved proteins were transferred to a nitrocellulose membrane .
the membrane was incubated with primary antibodies and then treated with horseradish peroxidase - conjugated anti - rabbit igg as a secondary antibody .
all bands were detected using an enhanced chemiluminescence system ( amersham biosciences , buckinghamshire , uk ) .
each set of experiments was done at least three times and results are presented as the mean  sd . the statistical significance of differences between mean values was assessed with student 's t - test or anova .
ang ii increases the intracellular ca concentration in vascular smooth muscle cells through a sequence of events following activation of ang ii type 1 receptor and l - type calcium channels [ 6 , 7 ] .
pe- or ang ii - induced contraction was significantly dilated by 53.6  7.8% and 66.0  5.4% , respectively , as compared to maximal tension ( figure 1 ) , indicating that that cre - mediated vasodilation may be related to decreased intracellular ca concentration . to determine the influence of cre on ca influx , the change of contraction was measured by adding cacl2 in an accumulative manner ( 0.3 , 0.6 , 1 , 1.5 , 2.5 , 5 , and 10  mm , in order ) before and after cre pretreatment in ca - free krebs solution containing 60  mm kcl .
the vasoconstriction rate at the aforementioned cacl2 concentrations were 7.7  8.1% , 23  16% , 44  16.7% , 59  17% , 72  14% , 88.4  9.4% , and 100% , respectively , ( the latter represents the maximum contraction value of the aortic ring , achieved at 10  mm cacl2 ) .
these contractions were significantly reduced by 1.9  2.2% , 5.8  4.1% , 8.3  6.3% , 11.4  4.4% , 22.3  1.9% , 24.8  6.2% , and 29.7  3.8% ( same respective cacl2 concentrations ) with cre - pretreatment ( figure 2 ) . to discern the effect of cre on the l - type calcium channel , the influence of the l - type calcium channel blocker nifedipine ( 100  m ) or verapamil ( 1  m ) , and the l - type calcium channel activator fpl64176 ( 10  m ) on vasorelaxation of cre against pe - induced contraction of aortic rings was measured .
pretreatment of aortic rings with nifedipine or verapamil significantly inhibited the relaxant effect of cre ( figure 3(a ) ) .
previous studies have shown that fpl64176 increases extracellular ca entry , thereby enhancing the cytosolic ca concentration [ 23 , 24 ] .
presently , fpl64176 induced contraction , which plateaued at 3.75  0.22  g in 30  min , was inhibited by 2.4  0.1  g with preincubation of cre ( figure 3(b ) ) . to assess whether cre is involved in ca release - mediated vasoconstriction from intracellular stores , the transient contraction by pe or caffeine was examined in cre preincubated aortic rings .
preincubation reduced the magnitude of contraction by pe from 0.32  0.6  g to 0.1  0.6  g ( figure 4(a ) ) . the transient contraction induced by 30  mm caffeine
was also reduced by cre pretreatment ( figure 4(b ) ) . to evaluate whether the relaxant effect of cre was involved in the plc pathway , the plc pathway inhibitor u73122 and activator m-3m3fbs were used .
u73122 pretreatment significantly inhibited the relaxant effect of cre on pe - induced contraction from 56.9  2.7% to 8.5  1.3% ( figure 5(a ) ) .
m-3m3fbs ( 5  g / ml)-induced contraction was relaxed significantly with cre treatment ( figure 5(b ) ) .
pi3k is directly activated by g - protein for generation of pip3 , which eventually stimulates l - type calcium channels in vascular myocytes [ 5 , 12 ] .
, ip3 stimulates intracellular ca release from the sr for vasoconstriction [ 15 , 17 ] .
presently , pe and m-3m3fbs increased the expression levels of pi3k ( 4.5  0.38 and 2.0  0.32 , resp . ) and pplc ( 4.1  0.30 and 2.5  0.14 , resp . ) , which were significantly decreased by cre . pe - induced pi3k expression was decreased by 2.8  0.70 at 50  g / ml and by 0.2  0.6 at 100  g / ml . pplc expression was also decreased by 2.0  0.30 at 50  g / ml and by 1.8  0.1 at 100  g / ml ( figure 6(a ) ) .
m-3m3fbs - induced pi3k expression was downregulated by 0.6  0.1 at 50  g / ml and by 0.5  0.2 at 100  g / ml , and pplc expression was decreased by 1.4  0.1 at 50  g / ml and by 0.3  0.1 at 100  g / ml ( figure 6(b ) ) .
its bark and twig are known as cinnamomi cortex ( cc ) and cinnamomi ramulus ( cr ) , respectively .
cc inhibits helicobacter pylori   and ameliorates sucrose - induced blood pressure elevation in spontaneously hypertensive rats .
furthermore , cre exerts an endothelium - independent vasodilatory response through inhibition of voltage - dependent ca channels .
the present study investigated the vasodilatory effect of cre resulting from the inhibition of both ca influx and release in rat aorta .  
pe or ang ii stimulate plc isoforms to generate ip3 through the activation of g proteins , causing release of activator ca from sr [ 9 , 10 , 1416 ] . presently , cre markedly and similarly relaxed aortic rings that were precontracted with pe or angii .
these results suggest that cre - mediated vasodilation may be involved in the regulation of ca mobilization . to assess this ,
firstly , whether cre actually inhibits extracellular ca influx or not , we measured the tension of aortic rings by accumulative addition of cacl2 in ca - free krebs solution containing 60  mm kcl .
preincubation with cre significantly reduced rat aortic contraction by addition of cacl2 , , indicating inhibition of ca influx .
nifedipine ( 100  m ) and verapamil ( 1  m ) pretreatment inhibited the vasodilative effect of cre on pe - induced constricted aortic rings from 54.9  4.1% to 15.3  2.1% and 13.9  5.2% , respectively .
in addition , preincubation with cre reduced the contraction of aortic ring by 10  m fpl64176 .
these results support the suggestion that the vasodilative effect of cre is related to the inhibition of l - type calcium channel in the cell membrane .  
the sr is the major source of ca release into the cytosol [ 1 , 16 , 17 ] .
this ca release is induced by the ip3 second messenger , which is generated by plc activation .
ca release from the sr is considered to be the initial mechanism in agonists such as pe- and ang ii - induced vasoconstriction [ 6 , 17 ] .
pe - induced transient constriction is dependent on ca release from the sr through the ip3 signal pathway in ca - free krebs solution ; however , caffeine is dependent on ca - induced ca release from the sr [ 27 , 28 ] . to demonstrate the effects of cre on ca release from the sr ,
transient contractions induced by pe in ca - free krebs solution and induced by caffeine in normal krebs solution were investigated .
cre significantly reduced the magnitudes of transient contraction by pe and caffeine , suggesting cre inhibits ca release from the sr by blocking the ip3-induced ca release and ca - induced ca release mechanisms .
pretreatment with the plc inhibitor u73122 significantly reduced the vasorelaxation of cre on pe - induced vasoconstriction , and cre relaxed m-3m3fbs - induced vasoconstriction .
additionally , we analyzed the expression levels of the intracellular signaling regulator proteins pi3k and plc .
pi3k generates various 3-phosphorylated phosphoinositides through activation by g - proteins , especially pi(3,4,5)p3 stimulates the l - type ca channel that plays an important role in the regulation of vascular tone [ 8 , 11 , 12 ] . on the other hand , plc formats the two potent second messengers ip3 and dag
especially , ip3 induces the activation of ip3 receptor on the sr membrane , opening a calcium channel , resulting in the release of ca into the cytosol [ 14 , 17 ] .
presently , pe- or m-3m3fbs - induced phosphorylation of plc and upregulation of pi3k / p85 protein expression were inhibited by cre ( figure 6 ) .
the collective data supports the idea that cre dilates vascular contraction through the inhibition of both ca influx via the l - type ca channel and ip3-induced ca release from the sr .   in conclusion
, the data supports the vasorelaxation of cre through the inhibition of ca influx and ca release .
therefore , cre may be useful as a drug for the treatment and prevention of high blood pressure associated with ca - dependent contraction of smooth muscle .
 contraction of vascular smooth muscle cells depends on the induction of cytosolic calcium ion ( ca2 + ) due to either ca2 + influx through voltage - gated ca2 + channels or to receptor - mediated ca2 + release from the sarcoplasmic reticulum . 
 the present study investigated the vasorelaxation effect of cinnamomi ramulus ethanol extract ( cre ) and the possible mechanisms in rat aorta . 
 cre ( 0.1  mg / ml ) relaxed vasoconstriction induced by phenylephrine ( pe ; 1  m ) and angiotensin ii ( 5  m ) . 
 preincubation with cre significantly reduced the rat aortic contraction by addition of cacl2 in ca2 + -free krebs solution and fpl64176 ( 10  m ) . 
 pretreatment with nifedipine ( 100  m ) or verapamil ( 1  m ) significantly reduced the cre - mediated vasorelaxation of pe - induced vascular contraction . 
 in addition , cre also relaxed the vascular contraction caused by m-3m3fbs ( 5  g / ml ) , but u73122 ( 10  m ) significantly inhibited the vasorelaxation of pe precontracted aortic rings . 
 furthermore , cre significantly reduced the magnitude of pe- and caffeine ( 30  mm)-induced transient contraction . in vascular strips , 
 cre downregulated the expression levels of phosphorylated plc and phosphoinositide 3-kinase elevated by pe or m-3m3fbs . 
 these results suggest that cre relaxes vascular smooth muscle through the inhibition of both ca2 + influx via l - type ca2 + channel and inositol triphosphate - induced ca2 + release from the sarcoplasmic reticulum . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion and Conclusions