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Vasodilatory action mechanisms of apigenin isolated from Apium graveolens in rat thoracic aorta
Biochimica et Biol~hY,~ic-a ..tcta. I t 15 ¢,i 99 i ) hr./ -- 74 :t-', It}01 Elsevier Science Publisher,. B.V. All rights rc',c~¢t] 1t31|4-4165/~1 '51~3.51)
69
B B A G E N 23612
Vasodilatory action mechanisms of apigenin :,solated from Apium grat,eolens in rat thoracic aorta F c n g - N i e n Ko, T u r - F u H u a n g a n d C h c - M i n g T e n g t)harmac~bJ,f.,wat hmittae, (".liege ~ t .~h'dictnt'. .\'at iomtl -lht. an ~ "nit cr~ity. Taipei ( Taitvan )
t Received lt~ April l~)t~I
Key words: Vast~iclaxatitm: ,,\9igcnin: ('alcium influx: (.-I .er.~,'oh'n.~ ~: ( R a l aorlaD
The effect of apigenin, isolated from Apium grareolcns, on the contraction of rat thoracic aorta was studied. Apigenin inhibited the contraction of aortic rings caused by cumulative concentrations of calcium (0.03-3 raM) in high potassium (60 raM) medium, with an IC~ o of about 48 /z M. Alter pretreatment it also inhibited norepinephrine (NE, 3 # M ) - i n d u c e d phasic and tonic contraction in a concentration (35-140/.tM)-dependent manner with an IC~o of 63 # M . At the plateau of NE-induced tonic contraction, addition of apigenin caused relaxation. This relaxing effect of apigenin was not antagonized by indomethacin t20 ttM) or methylene blue 150 ~ M ) , and still existed in endothelial denuded rat aorta or in the presence of nifedipine 12-100 # M ) . Neither cAMP nor cGMP levels were changed by apigenin. Both the formation of inositol monophosphate caused by NE and the phasic contraction induced by caffeine in the Ca-" +-free solution were unaffected by apigenin. 4SCa-"+ influx caused by either NE or K + was inhibited by apigenin concentration-dependently. It is concluded that apigenin relaxes rat thoracic aorta mainly by suppressing the Ca-" + influx through both voltage- and receptor-operated calcium channels.
Introduction
Flavonoids and biflavonoids are potent inhibitors o[ cyclic nucleotidc phosphodicsterasc (EC 3.1.4.17) in human and many animal tissues [1-2] with ~dffercnt selectivity and potency. Inhibition of cAMP phosphodiesterase has been implied in some pharmacological actions of flavonoids, such as inhibition of platelct aggregation and secretion [3-5]. However, this is not enough to imply directly the inhibition of phosphodiesterase in the mechanism of their pharmacological actions on all tissues. Celery (Apium grat'eoh'ns) is a common folk medicine used in China as an antihypertensive and antiinflammatory agent [6.7]. Apiin has been isolated from this plant and apigenin, a 4',5,7-trihydroxyflavone, was obtained after hydrolysis of apiin [8]. We have reported that apigenin could inhibit rabbit platelet aggregation through suppression of thromboxane A , formation caused by aggregation iaducers [0].
In this paper, we report the vasorelaxation effect of apigenin on rat thoracic aorta and try to elucidate its mode of action(s), Materials and Methods
Ah~terials Apigcnin, norepinephrine, acetyleholine, 3-isobutyll-mcthylxanthine (IBMX), sodium nitroprusside, methylene blue, trichloroacetic acid, bovine serum albumin, EGTA, EDTA, Dowex-I (111t]-201] mesh: ×8, chloride) resin, mvo-inositol and forskotin were obtained from Sigma Chemicals. tSCa-'+, myo-[2-3H]inositol and cAMP and cGMP R1A kits were purchased from Amersham. If drugs were dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO in the bathing solution did not exceed 0.1% and has no effect on the muscle contraction.
Mechank'al response Abbreviations: NE. n o r e p i n e p h r i n e : A C h . ac.qylcholine. C o r r e s p o n d e n c e : C.M. Tong. Pharmltcok~gical Institute. College of Medicine. National Taiwan University. No. I. Jen-Ai Rd.. Sect. I.
Taipei, Taiwan.
Wistar rats of either sex weighing 250 to 300 g were killed by a blow to the head. The thoracic aorta was isolated and excess fat and connective tissue were removed. Vessels were cut into rings of about 5 mm in !ength and mounted in organ baths containing 5 mi of Krebs solution of the following composition (mM):
70 NaC1 118,2, KCt 4.7, CaCI 2 1.9, MgSO 4 1.2, KH2PO 4 1.2, NaHCO 3 25 and glucose !1,7, The tissue bath solution was maintained at 37 °C and bubbled with a 95% O, and 5 ~ CO~ mixturc. Two stainless steel hooks were inserted into the aortic lumen, one was fixed, while the other was connected to a transducer. Aortac were equilibrated in the medium for 90 min with thrcc changes of Krebs solution and maintained under an optimal tension of 1 g before specific experimental protocols were initiated. Contractions were rccordcd isometrically via a force-displacement transducer connected to a GouId polygraph (Model 2400). In some experiments, the cndothetium was removed by rubbing with a cotton ball, and the ~,bscncc of acctylcholine-induced rchtxation was taken as an indicator that vessels wcrc denuded succcssfully. The contractile effects of calcium wcrc studied in rings stabilized in high K' solution without Ca 2+. Calcium was then added from stock dilutions to obtain the dcsired conccntraticms, and the effect of each Ca:* concentration was recorded. The maximal tension attaincd at 3 mM Ca:" was considered as 111t)'74. The high K* solution was prepared by substituting NaCI with KCt (60 raM) in an cquimolar amount.
cAMP assay of rat aorta The content of cAMV was assayed on aortic rings as described by Kauffrnan et ill. [1()]. After incubation of aortic rings with IMBX (i0 ~M) liar 5 rain, DMSO, forskolin or apigenin was added and incubatcd for another 5 rain. At the end of the reaction, the aortic rings wcre rapidly frozen in liquid nitrogen and stored at - 8 0 ° C until homogenized in 0.5 ml of 1(1% trichloroacetic acid using a Potter glass/glass homogenizer. The homogenate was centrifuged at 10000 × g lk)r 5 min and the supernatant was removcd and extracted four times with 3 vol. of diethyl ether, and the cAMP content was then assayed using RIA kits. The precipitate was used for protein assay [11]. cAMP ievcls werc cxpresscd as pmoi/mg protein.
cGMP assay of rat aorta Arotic rings prepared as described above, but not put under tension, were placed in l mi of Krebs solution for 1 h with 95% O, and 5% CO, at 37 ~ C. After incubation of aortic rings with the DMSO (0.1%, control), sodium nitroprusside or apigenin for 5 rain, the reaction was stopped by immersing the tissue into liquid nitrogen and storagc at - 8 0 ° C up to the time of thawing in 1(1% trichloroacetic acid and 4 mM EDTA. Aftcr homogenization with a Potter glass/glass homogenizer for 2-3 rain, the homogenate was centrifuged at 10000 × g for 5 rain. The cGMP content of supernatant, after extraction with diethyl ether for four times, was assayed using R1A kits [12], while the pre-
cipitate was used for protein assay [11]. The cGMP levels were expressed as pmol/mg protein.
Measurement of [ SH/inositol monophosphate The same procedure as described by Hirata et ai. [13] was used. Briefly, rat thoracic aortae were exposed to Krebs solution containing 10/xCi/ml of [3H]inositol for 3 h and gassed with 95% O~ and 5% CO_, mixture. The tissues were then transferred to tubes containing fresh Krebs solution with DMSO or apigenin for 5 min and saline or NE (3/2 M) was added and incubated for another 15 min. LiCI (10 raM) was added 5 min before NE to inhibit inositol monophosphatasc [14]. Aortae were then frozen in liquid nitrogen and homogenized in 1,3 ml of 10% trichioroacetic acid. After centrifugation, l ml of supernatant was collected and trichloroacetic acid was removed by washing four times with 3 vol. of diethyl ether. The inositol monophosphate in the aqueous phase was analyzed by application of sample to 1 ml of Dowex-I ion-exchange column according to the method of Neylon and Summers [15]. The tissue pellets were resuspended in 1.0 M NaOH and assayed for protein according to the method of Lowry et al. it l]. 45Ca 2 + itlflllX ~SCa2+ influx was measured in a manner similar to that described by Kaushik et al. [16]. Aortic rings were placed in test tubes containing Krebs solution with 2 p, Ci/mi of 45Ca-'+ in the presence of DMSO (0.1%) or apigenin and incubated for 5 min. NE (3 /.tM) or K + (60 raM) was then added and incubated for another 15 rain. After the incubation period, the tissues were transferred into test tubes containing 2 ml of ice-cold Ca 2+-free Krebs solution with 2 mM EGTA for 40 rain in order to remove extracellular "~~Ca-'L The tissues were thcn removed, lightly blotted with No. 5 Whatman filler paper, weighed and dissolved in 37% perchloric acid ((I.1 ml) at 75°C. The radioactivity was counted in a liquid scintillation counter (Packard Model 221){) CA). The concentration of drugs were expressed as final bath concentration. Results are expressed or plotted as the means _+ S.E. Student's t-test was used for statistical analysis. P values of less than 0.05 were considered to be significant. Results
Effects of apigenin on NE.in&tced phasic and tonic contractions In response to 3/.tM norepinephrine (NE), rat aorta generated a phasic followed by a tonic contraction maintained for at least 30 min. A more than 70% relaxation caused by acetylcholine (ACh, 3 p.M) indicated that endothelium in this preparation was intact,
[::Z] NE
PT-A Apigenin (70 ~zM) +NE gx~ Apigemn (140 ~M) + N~
120 N-"VqApigenin (35/~M) + NE
~
100 80-
"~ 60. O 40
0
Phv,~ic
Tonic
Fig. 1. Effects of apigenin on the phasic and tonic contractionn induced by NE in rat aorta, Various eon¢entrations of ~lpigenin or
DMSO (0.1%)were added 5 rain before the addition of NE (3 tiM) to induce muscle contraction. The contraction induced by NE ill the presence of D M S O was considered as I(}llr;, Values are express*cd as means +_S.E. of six experiments. * P < 0.1)5 and * "~ P < ().11()!, as compared with the respective control.
Pretreatment with apigcnin, 35 to 140 # M . inhibitcd the NE-induced phasic and tonic contraction in a concentration-dependent manner (Fig. 1), After 5 rain pretreatmcnt, the IC5, of apigenin on NE-induced tonic contraction was b3 p.M. If apigenin was added at the state of tonic contraction (10 rain after exposure to NE), a relaxation could
Effects of apige, tin on ttigh K ~-i,tduced cak'it,,,t-thTen"otttr~l('tiot!
dellt
In Ca ? ~-t'ree Krcbs solution containing 60 mM K ~'. the cumulativc addition of C a : " (0.03 to 3 raM) caused a stepwise increase of contraction in the rat aorta. After prctreatmcnt for 5 min, apigcnin (35 to 140 t.tM) a,
A, Intact Aorta
0.75 +, 0.02 g
be observed (Fig. 2A). The relaxations were 34.6 + 2.6. 55.2 +_ 13) and c)I.5 ___4.4%. for apigenin at 35, 70 and 14(1 # M . rcspcctively. The relaxing action of apigenin was not affected by indomethaci, (21)/,tM) or methylene bluc (50 /zM) which was added 3 rain before apigenin (data not shown). After removat of the endothelium from the rat aorta, the relaxing action of ACh was completely abolished, while apigenin still relaxed the denuded aorta, although the potency was somc~hat less than that in the intact aorta (Fig. 2B). By treating the denuded rat aorta with 2 ;tM nifedipinc for 15 rain. high K" (80 mM)-induced contraction was completely blocked (data not shown). Apigenin relaxed the nifcdipine (2 /zM)-treated aorta in a concentration dependent manner (Fig. 3b, c and d). If the concentration of nifedipine was increased to 20 or tOO ~ M , NE-induced tonic contracUon was inhibited progressively and the remaining tension was still relaxed by apigcnin (Fig. 3c and D.
Sift
Relaxation(%)
S ~ . - t :
2.R%
t ACh
DMSO
j,
1
A NE
35 b.
_f
0,76 + 0.02 g
1
_J
.......
i
55.2 J: 1.9%
-------
70
0,72 5 : 0 . 0 2 g
t
1 g
l
35
5 Min e,
C.
i
140
A
B. Denuded Aorta
~g[
70
d.
l
140
l
140
f.
5 Min 0 . 7 6 -2:0,06 g
140 i
NE
140 NE A p i g e n i n ( g M ) i
Fig. 2, Relaxation of NE-induced tonic contraction caused by apigenin in intact (panel A) and denuded (panel B) aorta. D M S O (0.t%) or apigenin was added It} rain after the exposure of aorta to
NE (3 gM). The g relaxation seen 10 min later was calculated and expressed as the means+S.E, of five to six experiments. Acetylcholine (ACh. 3/.tM) was used to determine whether the endothelium was intact or denuded.
NE Apigenin (,uM)
NE
Apigenin (~uM) apigeninon NE-inducedtonic contr;Ic-
Fig, 3. Relaxationeffect 0f tion in the presence of nifcdipine, After pretreatment of denuded rat aorta with D M S O (0. lC;-) or nifedipine (2 # M for b, c. d" _.2(}or [{~(I /zM for e and f) for 15 rain, NE (3 # M ) was used to induce muscle contraction. D M S O (0.1c~)or apigenin was added It) rain after the exposure of aorta It) NE to cause the relaxalion. Acetylcholine (ACh, 3 ta.M) wa,,, used to determine whether the endothe[ium was int;,ct or denuded.
72 Apigenin (~M) 0
too l 9oq
TABLE Ii
Effect of apigenin on the [iH]inositol monophosphate formation of rat thoracic aorta
i
soj
•~ 704
3s
"~
[
~
r 140
0O~ 6 0 50. O
40. 30.
z0
After incubation of [3H]inositol-tabeled rat aorta with DMSO (0.1ek, resting and control) or apigenin (140 # M ) for 5 min, saline (resting) or NE was added and incubation for another 15 rain. The reaction was terminated and the [3H]inositol monophosphate was separated, collected and counted by liquid scintillation counter. Results are expressed as the means _+S.E. (n) Treatment
• ~
0.03
0.10
.
.
.
0.30
Calcium
.
1.00
3.00
(mM)
Fig. 4. Effects of ;,pigcnin on the ('a ~"'-dependent contraction of rat aorta induced by high K ' (6(I mMl. Aorta was Dreineubaled with 0.1% DMSO ( ~ l or apigenin {35 #M, o: 70 p.M. ,',; 140 p.M, A ) a t 37°C for 5 n]in. then cumuh, tive concentrations of Ca 2' (0.03-3 mM) were used to trigger the contraction. Each point represents the me:ms_+S.E, of six determinations. All control data point were significantly inhibilcd hy various concentrations of apigenin.
[:~H]inositol monophosphate
(c.p.m./mg protein) Resting NE (3 #M) Apigenin (140 # M ) + NE
1091+_ 85(4) } p < o . o o l 2352+211 (4) 2417_+231 (4) } p > o . 0 5
Effects o f apigenin on c A M P and c G M P formation Cyclic nucleotides sured
by
contents
radioimmunoassay.
of rat aorta were meaAs
shown
in T a b l e
1,
A.
50
inhibited this contr;tction in a concentration-dependent manner with an ICs, of about 48 # M for Ca -'+ concentration of i rnM (Fig. 4).
i 40
Nz (3~M) Apigenin (35/~M) + NE EZ~ Apigenin (70 p.M) + NE ~X~ Apigenin (140 p,M)+NE
-i--
+
*0
+
:30.
Effects o f apigenin on caff,,ine-induced contraction After
equilibrium
in t h e C a 2 + - f r e e
Krebs solution
'~
Ill
~.o-
for 5 min, caffeine (10 raM) caused a rapid phasic c o n t r a c t i o n o f r a t a o r t a ( 0 . 4 0 + 0.03 g, n = 4). A p i g e n i n
(140 g M ) pretreatment for 5 rain did not affect this contraction (0.38 + 0.03 g, n = 4). Nifedipine (2 # M ) also had no effect (0.38 + 0.03 g, n = 4), while procaine (10 raM) completely inhibited the caffeine-induced
.
phasic contraction. 6O
~, TABLE I
4-
Effi'cts of apigenin on the cGMP and cAM/' formation of rat aorta After preincubation of aortic rings with (for cAMP) or without (for cGMP) IBMX (10 btMl for 15 min, DMSO (0.1'7~;, control), sodium nitroprusside, forskolin or apigenin was added for another 5 rain .'rod the reaction was stopped by immersing the tissue into liquid nitrogen. cGMP and cAMP contents in ral aorta were measured by radioimmunoassay. Results are expressed as the means+S.E. (n = 5-7). * * * P < 0.(}[ll as compared with Ihe respective control. Treatment
cGMP
cAMP
(pmol/mg protein) Control Sodium nitroprusside (l(I ~M) Forskolin (10 ~M) Apigenin (35 p.M) (70 p.M) (370 IzM)
2.2 +_0.22 6.8(l+0.55 * * *
2.23 ± (l,28 -
-
28.57_+5.17 *** 2.12±0.22 2.55 + 0.15 4.63_+ 0,5 * " *
2.43 + 0.35
+
50 40
d, ao
/-
r-a ZCi (e0 raM) , ~
Apigenin (35 ~M) + KCI Apigenir, (70 t~M) + KC1 Apigenin (140 ~M) + KCI
®
~d 20
0
k\\\\\'i
Fig. 5. Effects of apigenin on the 45Ca-" + influx induced by NE and KC[. Aortic rings were placed in test tubes containing Krebs solution with 4"~Ca2" (2 AtCi/mL) and incubated for 5 rain with DMSO (||.1%) or various concentrations of apigenin, then NE (3 p.M, panel A), KCI (60 raM, panel B) or saline [resting) was added and incubated for another 15 rain. 45CaZ+ influx into the muscle was measured. The data a,e expressed as percent increase in 45Ca2" uptake over the resting value. The values are expressed as means + S.E. of four to five experiments. * P
73 sodium nitroprusside and tk~rskolin markedly elevated cGMP and cAMP levels in rat aorta, respectively. cAMP level was not affected by apigcnin. The cGMP level was also not elevated by apigcnin unless tb.c concentration of 370/z M was used (Table i).
Effects of apigeni, on NE-imhwed inositol monophosphate formation Exposure of rat aorta to 3/.tM NE for 15 ,nin in the presence of 1(I mM LiC! clcvatcd inositol monophosphate levels. Prctrcatmen: with apigcnin (140/.tM) l\~r 5 min did not inhibit the inositol monophosphatc tt~rmation caused by NE (Table I!). Effects of apigenin on 4~Ca2 ~ inJlltr bzdm'cd by NE or high K ÷ in the presence of NE (3 /zML the ~ ( ' a 2" influx was increased 41.8 + 1.9% over that in the control rings. This increase of "~SCa-'~ influx was inhibited by apigenin (35 to 140 # M ) in a concentration dependent manner (Fig. 5A). A similar inhibitory, effect on high K~'-induced 4SCa2" influx was also shown in Fig. 5B. High K ÷ (60 mM) caused a 51.4 +_ 3.(Jeff, increase of 45Ca2+ influx over that in the control rings, and this increase was also inhibited by apigcnin (35 to 140 txM) in a concentration dependent manner. Discussion
Vascular endothelium plays an important role in controlling the vascular tone via secretion cff both relaxant and contractile factors [17.18]. In response to a variety of neurohumoral and physical stimuli, endothelial cells release endothelium-dcpendent wlsodilators such as endothelium-dcrived relaxing factor (EDRF) and prostacyclin (PGI_,). The relaxing action of apigenin persisted in denuded or intact aorta in the presence of indomethacin (21)/.tM) or methylene bluc (50/zM). Indomethacin was known to block the generation of PGI.,, while methylene blue was reported to inhibit the E D R F activation of guanylatc cyclase [191~ Thus, the vasorelaxation caused by apigcnin was independent of the endothelium and was not mediated by either E D R F or PGI ~. Cyclic nucleotides are very important for relaxing vascular smooth muscle, cGMP causes Ca 2 + extrussion or sequestration [20,21] and inhibits Ca :~ uptake [22], contractile elements [23] or receptor-linked phosph0inositides breakdown [24,25]. cAMP can dilate vascular smooth muscle either by phosphorylation or" myosin light chain kinase [26,27], or increasing calcium uptake by the sarcoplasmic reticulum [28], or acting in other ways to reduce free cytosolic calcium [29]. It has been reported that flavonoids and biflavonoids are potent inhibitors of cyclic nucleotide phosphodiesterase [I,2], and may play roles in inhibition of platelct aggregation
and secretion [3-5]. Neither cAMP nor cGMP levels wcrc increased by apigenin at concentrations causing significant relaxation. This implies that the relaxing ,'ff,~ct of upigcnin is not duc to the formation of cyclic nuclcotidcs. Apigcnin inhibited the phasic contraction caused by NE. It has been reported that activation of o¢~-adrenergic receptors by NE stimulates phosphoinositides turnover to increase the concentration of inositot trisphosphatc and subsequently release the cellular ('a:" to mediate the phasic contraction [311,31]. However, NE-induccd inositol monophosphatc formation was nt~t inhibited by apigenin. This rules out the possibility that apigcnin may inhibit the NE-induced phasic contraction through inhibition of NE-induccd phosphoinositides hydrolysis. Apigenin also had no effect on caffeine-induced phasic contraction. It is now generally accepted that caffeine can release intracellular Ca: ~ in vascular smooth muscle and that there is an twcrlap between the NE- and caffeine-sensitive Ca 2~ stores [32-34]. Thcretorc, this result may imply that apigenin does not inhibit the release of intracellular Ca 2" from caffeine-sensitive as wctl as NE-scnsitive Ca 2 ~ stores. However. the action mechanism(s) of apigenin on the inhibition of NE-induced phasic contraction is not clear now, it may act on the site(s) after Ca 2+ was released. it is well known that high K'-induced contraction in smooth muscle is mediated by an increase in Ca-'* influx through voltage-gated Ca-"" channels [35]. Sincc apigenin inhibited Ca2%depcndent contraction and t~Ca2 ~ influx in high K ~ medium, it maybe a blockcr of voltage-gated Ca 2 ~ channel. The maintenance of tension (tonic contraction) in response to NE results primary from Ca -'~ entry through receptor-operated Ca-" channels with little requirement for Ca-' + entry through voltage-gated Ca-'" channel [36.37]. The NE-induced tonic contraction and "~SCa2 ~ influx were both inhibited by apigenin (Figs. ! and 5A). Furthermore, apigenin still relaxed the NEinduced tonic tension in a concentration dependent manner, in the presence of nifedipine (2 /zM, Fig. 3), which completely blocked high K + (80 mM)-induced contraction, in general, the membrane potential of vascular smooth muscle cell depolarized by NE was not more than 20 mV [38-40], and the increase of extracellular K ~ from 4.7 to 60 mM depolarized the vascular membrane potential about 60 mV [41] and about thc same value calculated as from Nernst equation assuming the intracellular K ~ is t50 mM. Thus, this indicates that apigcnin maybe a blocker of receptor-operated Ca 2 " influx. Rat aorta is different to rabbit aorta in some of its properties. In rabbit aorta, the voltage-gated Ca -'* channel and receptor-operated Ca z+ channel are independent. The former is activated by membrane depolarization and is more selectively blocked by organic
74 C a z+ c h a n n e l b l o c k e r s such as v e r a p a m i l , n i f e d i p i n e and diltiazem, w h e r e a s the latter is a c t i v a t e d by t h e b i n d i n g o f agonist to their r e c e p t o r s and is m o r e selectively b l o c k e d by s o d i u m n i t r o p r u s s i d e [22,42]. H o w -
ever, these two types of Ca z+ channels are functionally not completely separated in rat aorta [35,36], and voltage-gated Ca -'+ channel blocker (e.g., nitrendipine, nifedipine and verapamil) usually inhibited NE-induced contraction completely in higher concentrations [43]. Thus, in rat aorta, both of voltage-gated and r e c e p t o r - o p e r a t e d Ca 2+ channels are not totally separated and inhibited by apigenin. If apigenin is a selective inhibitor of receptor or voltage operated Ca 2+ channel in rabbit aorta needs further experiments to find out. It is concluded that the inhibitory effect of apigenin on thc c o n t r a c t i l e r e s p o n s e c a u s e d by high K + a n d N E in rat t h o r a c i c a o r t a is mainly d u e to inhibition o f Ca 2+
influx through both voltage- and receptor-operated Ca 2+ channels.
Acknowledgements T h i s w o r k was s u p p o r t e d by r e s e a r c h g r a n t s o f t h e N a t i o n a l S c i e n c e C o u n c i l of the R e p u b l i c o f C h i n a
(NSC79-0412-B002-26) and the National Research Institute of Chinese Medicine.
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11 Lowry, D.H., Rosebrough, N.J., Farr. A.L and Randall. R.J. (1951) J. Biol. Chem. 193, 265-275. 12 hoh, T., Izumi, H. and Kuriyama, H. (1982) J. Physiol. 32~, 475-493. 13 Hirata, M., Kohse, K.P., Chang, C.H., |keb, T. and Murad, F. (1990) J. Biol. Chem. 265, 1268-1273. 14 Berridge, M.J., Downes, C.P. and Hanley. M.R. (1982) Biochem. J. 206, 587-595. 15 Neylon, C.B. and Summers, R.J. (1987) Br. J. Pharmacol. 91: 367-376. 16 Kaushik, D.M., Roger. F.P. and Van Breemen, C. (1980) Eur. J. Pharmacol. 61. 159-165. 17 Jaffe, E.A. (1985) Ann. NY Acad. Sci. 454, 279-291. 18 Vanhoutte, P,M., Rubanyi, G.M., Miller, V.M. and Houston, D.S. (1986) Annu. Rev. Physiol, 48, 3[)7-330. 19 Ignarro, LJ. (1989) FASEB J. 3, 31-36. 20 Zsoter, TT., Henein, N.F. and Wolchinsky, C. (1977) Eur. J. Pharmacol. 45, 7-12. 21 Popescu. L.M., Panoiu, C., Hinescu. M. and Nato, U. (1985) Eur. J. Pharmacoi. 107, 393-394. 22 Karaki, H., Nakagawa, H. and Urakawa, N. (1984) Br. J. Pharmacol. 81,393-394. 23 Pfitzer, G., Merkel. L., Ruegg, J.G. and i[ofmann, F. (1986) Pflugers Arch. 4{)7, 87-91. 24 Rapoport, R.M. (1986) Circ. Res. 58, 407-410. 25 Lang. D. and Lewis, M.J. (1989)J. Physiol (Lond.)441, 45-52. 26 Adelsteim R.S. and Eistenherg, E. (1980) Annu. Rev. Biochem. 49, 921-956. 27 Silver. P.J. and Disaivo, J. (1979) J. Biol, Chem. 254, 9951-9934. 28 Seheid. C.R., Honeyman, T.W. and Fay, F.S, (1979) Nature (Lond.) 277, 32-36. 29 Kam,n, K.E and Stult, J.T. (1985) Ann. Rev, Pharmacol. Toxieol. 25, 593-620. 30 Hashimoto, T.M., Hirata, H., Itoh, H., Kanmura, Y. and Kuriyama, H. (1986)J. Physiol. (Lond,) 370, 605-618. 31 Suematsu, E., Hirata, M. and Kuriyama, H. (1984) Biochem. Biophys. Res, Commun. 120, 481-485. 32 Saida, K. and Van Breemen, C. (1984) Nature (Lond.) 281, 682-083. 33 ltoh, T., lzumi, H. and Kuriyama, H, (1983) J. Physiol. 337. 6(19-629. 34 Leijten, P.A.A, and Van Breemen, C. (1989) J. Physiol. (Loud.) 357, 327-339. 35 Karaki, H, and Weir, G.B. (1984) Gastroenterology 87, 960-970, 36 Bolton, T.B. (1979} Physiol. Rev. 59, 607-618, 37 Weiss, G.B. (t983) in Ca -`+ Enlry Blockers, Adenosine and Neurohumors (Merrill, G,F. and Weiss, H.R.. eds.), pp. 63-72. Urban and Sehwarzenbery, Baltimore. 38 Mekata, F. (1974)J. Physiol. (Lond.) 242, 143-155. 39 Mekala, F. (1979)J. Physiol. (Lond.) 293, 11-21. 40 Wallnofer, A., Weir, S., Riiegg, U. and Cauvin, C, (1989) J. Cardiovasc. Pharmacol. 13 (Suppl. 5), S23-$31. 41 Hermsmeyer, K, (1976) Circ. Res. 38, 362-366. 42 lip, K,, Karaki. H. and Urakawa, N. (1977) Eur. J. Pharmacol. 46, 9-14, 43 Godfraind, T., Miller, R. and Wibo, M. (1986) Pharmacol. Rev, 38, 321-416.
69
B B A G E N 23612
Vasodilatory action mechanisms of apigenin :,solated from Apium grat,eolens in rat thoracic aorta F c n g - N i e n Ko, T u r - F u H u a n g a n d C h c - M i n g T e n g t)harmac~bJ,f.,wat hmittae, (".liege ~ t .~h'dictnt'. .\'at iomtl -lht. an ~ "nit cr~ity. Taipei ( Taitvan )
t Received lt~ April l~)t~I
Key words: Vast~iclaxatitm: ,,\9igcnin: ('alcium influx: (.-I .er.~,'oh'n.~ ~: ( R a l aorlaD
The effect of apigenin, isolated from Apium grareolcns, on the contraction of rat thoracic aorta was studied. Apigenin inhibited the contraction of aortic rings caused by cumulative concentrations of calcium (0.03-3 raM) in high potassium (60 raM) medium, with an IC~ o of about 48 /z M. Alter pretreatment it also inhibited norepinephrine (NE, 3 # M ) - i n d u c e d phasic and tonic contraction in a concentration (35-140/.tM)-dependent manner with an IC~o of 63 # M . At the plateau of NE-induced tonic contraction, addition of apigenin caused relaxation. This relaxing effect of apigenin was not antagonized by indomethacin t20 ttM) or methylene blue 150 ~ M ) , and still existed in endothelial denuded rat aorta or in the presence of nifedipine 12-100 # M ) . Neither cAMP nor cGMP levels were changed by apigenin. Both the formation of inositol monophosphate caused by NE and the phasic contraction induced by caffeine in the Ca-" +-free solution were unaffected by apigenin. 4SCa-"+ influx caused by either NE or K + was inhibited by apigenin concentration-dependently. It is concluded that apigenin relaxes rat thoracic aorta mainly by suppressing the Ca-" + influx through both voltage- and receptor-operated calcium channels.
Introduction
Flavonoids and biflavonoids are potent inhibitors o[ cyclic nucleotidc phosphodicsterasc (EC 3.1.4.17) in human and many animal tissues [1-2] with ~dffercnt selectivity and potency. Inhibition of cAMP phosphodiesterase has been implied in some pharmacological actions of flavonoids, such as inhibition of platelct aggregation and secretion [3-5]. However, this is not enough to imply directly the inhibition of phosphodiesterase in the mechanism of their pharmacological actions on all tissues. Celery (Apium grat'eoh'ns) is a common folk medicine used in China as an antihypertensive and antiinflammatory agent [6.7]. Apiin has been isolated from this plant and apigenin, a 4',5,7-trihydroxyflavone, was obtained after hydrolysis of apiin [8]. We have reported that apigenin could inhibit rabbit platelet aggregation through suppression of thromboxane A , formation caused by aggregation iaducers [0].
In this paper, we report the vasorelaxation effect of apigenin on rat thoracic aorta and try to elucidate its mode of action(s), Materials and Methods
Ah~terials Apigcnin, norepinephrine, acetyleholine, 3-isobutyll-mcthylxanthine (IBMX), sodium nitroprusside, methylene blue, trichloroacetic acid, bovine serum albumin, EGTA, EDTA, Dowex-I (111t]-201] mesh: ×8, chloride) resin, mvo-inositol and forskotin were obtained from Sigma Chemicals. tSCa-'+, myo-[2-3H]inositol and cAMP and cGMP R1A kits were purchased from Amersham. If drugs were dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO in the bathing solution did not exceed 0.1% and has no effect on the muscle contraction.
Mechank'al response Abbreviations: NE. n o r e p i n e p h r i n e : A C h . ac.qylcholine. C o r r e s p o n d e n c e : C.M. Tong. Pharmltcok~gical Institute. College of Medicine. National Taiwan University. No. I. Jen-Ai Rd.. Sect. I.
Taipei, Taiwan.
Wistar rats of either sex weighing 250 to 300 g were killed by a blow to the head. The thoracic aorta was isolated and excess fat and connective tissue were removed. Vessels were cut into rings of about 5 mm in !ength and mounted in organ baths containing 5 mi of Krebs solution of the following composition (mM):
70 NaC1 118,2, KCt 4.7, CaCI 2 1.9, MgSO 4 1.2, KH2PO 4 1.2, NaHCO 3 25 and glucose !1,7, The tissue bath solution was maintained at 37 °C and bubbled with a 95% O, and 5 ~ CO~ mixturc. Two stainless steel hooks were inserted into the aortic lumen, one was fixed, while the other was connected to a transducer. Aortac were equilibrated in the medium for 90 min with thrcc changes of Krebs solution and maintained under an optimal tension of 1 g before specific experimental protocols were initiated. Contractions were rccordcd isometrically via a force-displacement transducer connected to a GouId polygraph (Model 2400). In some experiments, the cndothetium was removed by rubbing with a cotton ball, and the ~,bscncc of acctylcholine-induced rchtxation was taken as an indicator that vessels wcrc denuded succcssfully. The contractile effects of calcium wcrc studied in rings stabilized in high K' solution without Ca 2+. Calcium was then added from stock dilutions to obtain the dcsired conccntraticms, and the effect of each Ca:* concentration was recorded. The maximal tension attaincd at 3 mM Ca:" was considered as 111t)'74. The high K* solution was prepared by substituting NaCI with KCt (60 raM) in an cquimolar amount.
cAMP assay of rat aorta The content of cAMV was assayed on aortic rings as described by Kauffrnan et ill. [1()]. After incubation of aortic rings with IMBX (i0 ~M) liar 5 rain, DMSO, forskolin or apigenin was added and incubatcd for another 5 rain. At the end of the reaction, the aortic rings wcre rapidly frozen in liquid nitrogen and stored at - 8 0 ° C until homogenized in 0.5 ml of 1(1% trichloroacetic acid using a Potter glass/glass homogenizer. The homogenate was centrifuged at 10000 × g lk)r 5 min and the supernatant was removcd and extracted four times with 3 vol. of diethyl ether, and the cAMP content was then assayed using RIA kits. The precipitate was used for protein assay [11]. cAMP ievcls werc cxpresscd as pmoi/mg protein.
cGMP assay of rat aorta Arotic rings prepared as described above, but not put under tension, were placed in l mi of Krebs solution for 1 h with 95% O, and 5% CO, at 37 ~ C. After incubation of aortic rings with the DMSO (0.1%, control), sodium nitroprusside or apigenin for 5 rain, the reaction was stopped by immersing the tissue into liquid nitrogen and storagc at - 8 0 ° C up to the time of thawing in 1(1% trichloroacetic acid and 4 mM EDTA. Aftcr homogenization with a Potter glass/glass homogenizer for 2-3 rain, the homogenate was centrifuged at 10000 × g for 5 rain. The cGMP content of supernatant, after extraction with diethyl ether for four times, was assayed using R1A kits [12], while the pre-
cipitate was used for protein assay [11]. The cGMP levels were expressed as pmol/mg protein.
Measurement of [ SH/inositol monophosphate The same procedure as described by Hirata et ai. [13] was used. Briefly, rat thoracic aortae were exposed to Krebs solution containing 10/xCi/ml of [3H]inositol for 3 h and gassed with 95% O~ and 5% CO_, mixture. The tissues were then transferred to tubes containing fresh Krebs solution with DMSO or apigenin for 5 min and saline or NE (3/2 M) was added and incubated for another 15 min. LiCI (10 raM) was added 5 min before NE to inhibit inositol monophosphatasc [14]. Aortae were then frozen in liquid nitrogen and homogenized in 1,3 ml of 10% trichioroacetic acid. After centrifugation, l ml of supernatant was collected and trichloroacetic acid was removed by washing four times with 3 vol. of diethyl ether. The inositol monophosphate in the aqueous phase was analyzed by application of sample to 1 ml of Dowex-I ion-exchange column according to the method of Neylon and Summers [15]. The tissue pellets were resuspended in 1.0 M NaOH and assayed for protein according to the method of Lowry et al. it l]. 45Ca 2 + itlflllX ~SCa2+ influx was measured in a manner similar to that described by Kaushik et al. [16]. Aortic rings were placed in test tubes containing Krebs solution with 2 p, Ci/mi of 45Ca-'+ in the presence of DMSO (0.1%) or apigenin and incubated for 5 min. NE (3 /.tM) or K + (60 raM) was then added and incubated for another 15 rain. After the incubation period, the tissues were transferred into test tubes containing 2 ml of ice-cold Ca 2+-free Krebs solution with 2 mM EGTA for 40 rain in order to remove extracellular "~~Ca-'L The tissues were thcn removed, lightly blotted with No. 5 Whatman filler paper, weighed and dissolved in 37% perchloric acid ((I.1 ml) at 75°C. The radioactivity was counted in a liquid scintillation counter (Packard Model 221){) CA). The concentration of drugs were expressed as final bath concentration. Results are expressed or plotted as the means _+ S.E. Student's t-test was used for statistical analysis. P values of less than 0.05 were considered to be significant. Results
Effects of apigenin on NE.in&tced phasic and tonic contractions In response to 3/.tM norepinephrine (NE), rat aorta generated a phasic followed by a tonic contraction maintained for at least 30 min. A more than 70% relaxation caused by acetylcholine (ACh, 3 p.M) indicated that endothelium in this preparation was intact,
[::Z] NE
PT-A Apigenin (70 ~zM) +NE gx~ Apigemn (140 ~M) + N~
120 N-"VqApigenin (35/~M) + NE
~
100 80-
"~ 60. O 40
0
Phv,~ic
Tonic
Fig. 1. Effects of apigenin on the phasic and tonic contractionn induced by NE in rat aorta, Various eon¢entrations of ~lpigenin or
DMSO (0.1%)were added 5 rain before the addition of NE (3 tiM) to induce muscle contraction. The contraction induced by NE ill the presence of D M S O was considered as I(}llr;, Values are express*cd as means +_S.E. of six experiments. * P < 0.1)5 and * "~ P < ().11()!, as compared with the respective control.
Pretreatment with apigcnin, 35 to 140 # M . inhibitcd the NE-induced phasic and tonic contraction in a concentration-dependent manner (Fig. 1), After 5 rain pretreatmcnt, the IC5, of apigenin on NE-induced tonic contraction was b3 p.M. If apigenin was added at the state of tonic contraction (10 rain after exposure to NE), a relaxation could
Effects of apige, tin on ttigh K ~-i,tduced cak'it,,,t-thTen"otttr~l('tiot!
dellt
In Ca ? ~-t'ree Krcbs solution containing 60 mM K ~'. the cumulativc addition of C a : " (0.03 to 3 raM) caused a stepwise increase of contraction in the rat aorta. After prctreatmcnt for 5 min, apigcnin (35 to 140 t.tM) a,
A, Intact Aorta
0.75 +, 0.02 g
be observed (Fig. 2A). The relaxations were 34.6 + 2.6. 55.2 +_ 13) and c)I.5 ___4.4%. for apigenin at 35, 70 and 14(1 # M . rcspcctively. The relaxing action of apigenin was not affected by indomethaci, (21)/,tM) or methylene bluc (50 /zM) which was added 3 rain before apigenin (data not shown). After removat of the endothelium from the rat aorta, the relaxing action of ACh was completely abolished, while apigenin still relaxed the denuded aorta, although the potency was somc~hat less than that in the intact aorta (Fig. 2B). By treating the denuded rat aorta with 2 ;tM nifedipinc for 15 rain. high K" (80 mM)-induced contraction was completely blocked (data not shown). Apigenin relaxed the nifcdipine (2 /zM)-treated aorta in a concentration dependent manner (Fig. 3b, c and d). If the concentration of nifedipine was increased to 20 or tOO ~ M , NE-induced tonic contracUon was inhibited progressively and the remaining tension was still relaxed by apigcnin (Fig. 3c and D.
Sift
Relaxation(%)
S ~ . - t :
2.R%
t ACh
DMSO
j,
1
A NE
35 b.
_f
0,76 + 0.02 g
1
_J
.......
i
55.2 J: 1.9%
-------
70
0,72 5 : 0 . 0 2 g
t
1 g
l
35
5 Min e,
C.
i
140
A
B. Denuded Aorta
~g[
70
d.
l
140
l
140
f.
5 Min 0 . 7 6 -2:0,06 g
140 i
NE
140 NE A p i g e n i n ( g M ) i
Fig. 2, Relaxation of NE-induced tonic contraction caused by apigenin in intact (panel A) and denuded (panel B) aorta. D M S O (0.t%) or apigenin was added It} rain after the exposure of aorta to
NE (3 gM). The g relaxation seen 10 min later was calculated and expressed as the means+S.E, of five to six experiments. Acetylcholine (ACh. 3/.tM) was used to determine whether the endothelium was intact or denuded.
NE Apigenin (,uM)
NE
Apigenin (~uM) apigeninon NE-inducedtonic contr;Ic-
Fig, 3. Relaxationeffect 0f tion in the presence of nifcdipine, After pretreatment of denuded rat aorta with D M S O (0. lC;-) or nifedipine (2 # M for b, c. d" _.2(}or [{~(I /zM for e and f) for 15 rain, NE (3 # M ) was used to induce muscle contraction. D M S O (0.1c~)or apigenin was added It) rain after the exposure of aorta It) NE to cause the relaxalion. Acetylcholine (ACh, 3 ta.M) wa,,, used to determine whether the endothe[ium was int;,ct or denuded.
72 Apigenin (~M) 0
too l 9oq
TABLE Ii
Effect of apigenin on the [iH]inositol monophosphate formation of rat thoracic aorta
i
soj
•~ 704
3s
"~
[
~
r 140
0O~ 6 0 50. O
40. 30.
z0
After incubation of [3H]inositol-tabeled rat aorta with DMSO (0.1ek, resting and control) or apigenin (140 # M ) for 5 min, saline (resting) or NE was added and incubation for another 15 rain. The reaction was terminated and the [3H]inositol monophosphate was separated, collected and counted by liquid scintillation counter. Results are expressed as the means _+S.E. (n) Treatment
• ~
0.03
0.10
.
.
.
0.30
Calcium
.
1.00
3.00
(mM)
Fig. 4. Effects of ;,pigcnin on the ('a ~"'-dependent contraction of rat aorta induced by high K ' (6(I mMl. Aorta was Dreineubaled with 0.1% DMSO ( ~ l or apigenin {35 #M, o: 70 p.M. ,',; 140 p.M, A ) a t 37°C for 5 n]in. then cumuh, tive concentrations of Ca 2' (0.03-3 mM) were used to trigger the contraction. Each point represents the me:ms_+S.E, of six determinations. All control data point were significantly inhibilcd hy various concentrations of apigenin.
[:~H]inositol monophosphate
(c.p.m./mg protein) Resting NE (3 #M) Apigenin (140 # M ) + NE
1091+_ 85(4) } p < o . o o l 2352+211 (4) 2417_+231 (4) } p > o . 0 5
Effects o f apigenin on c A M P and c G M P formation Cyclic nucleotides sured
by
contents
radioimmunoassay.
of rat aorta were meaAs
shown
in T a b l e
1,
A.
50
inhibited this contr;tction in a concentration-dependent manner with an ICs, of about 48 # M for Ca -'+ concentration of i rnM (Fig. 4).
i 40
Nz (3~M) Apigenin (35/~M) + NE EZ~ Apigenin (70 p.M) + NE ~X~ Apigenin (140 p,M)+NE
-i--
+
*0
+
:30.
Effects o f apigenin on caff,,ine-induced contraction After
equilibrium
in t h e C a 2 + - f r e e
Krebs solution
'~
Ill
~.o-
for 5 min, caffeine (10 raM) caused a rapid phasic c o n t r a c t i o n o f r a t a o r t a ( 0 . 4 0 + 0.03 g, n = 4). A p i g e n i n
(140 g M ) pretreatment for 5 rain did not affect this contraction (0.38 + 0.03 g, n = 4). Nifedipine (2 # M ) also had no effect (0.38 + 0.03 g, n = 4), while procaine (10 raM) completely inhibited the caffeine-induced
.
phasic contraction. 6O
~, TABLE I
4-
Effi'cts of apigenin on the cGMP and cAM/' formation of rat aorta After preincubation of aortic rings with (for cAMP) or without (for cGMP) IBMX (10 btMl for 15 min, DMSO (0.1'7~;, control), sodium nitroprusside, forskolin or apigenin was added for another 5 rain .'rod the reaction was stopped by immersing the tissue into liquid nitrogen. cGMP and cAMP contents in ral aorta were measured by radioimmunoassay. Results are expressed as the means+S.E. (n = 5-7). * * * P < 0.(}[ll as compared with Ihe respective control. Treatment
cGMP
cAMP
(pmol/mg protein) Control Sodium nitroprusside (l(I ~M) Forskolin (10 ~M) Apigenin (35 p.M) (70 p.M) (370 IzM)
2.2 +_0.22 6.8(l+0.55 * * *
2.23 ± (l,28 -
-
28.57_+5.17 *** 2.12±0.22 2.55 + 0.15 4.63_+ 0,5 * " *
2.43 + 0.35
+
50 40
d, ao
/-
r-a ZCi (e0 raM) , ~
Apigenin (35 ~M) + KCI Apigenir, (70 t~M) + KC1 Apigenin (140 ~M) + KCI
®
~d 20
0
k\\\\\'i
Fig. 5. Effects of apigenin on the 45Ca-" + influx induced by NE and KC[. Aortic rings were placed in test tubes containing Krebs solution with 4"~Ca2" (2 AtCi/mL) and incubated for 5 rain with DMSO (||.1%) or various concentrations of apigenin, then NE (3 p.M, panel A), KCI (60 raM, panel B) or saline [resting) was added and incubated for another 15 rain. 45CaZ+ influx into the muscle was measured. The data a,e expressed as percent increase in 45Ca2" uptake over the resting value. The values are expressed as means + S.E. of four to five experiments. * P
73 sodium nitroprusside and tk~rskolin markedly elevated cGMP and cAMP levels in rat aorta, respectively. cAMP level was not affected by apigcnin. The cGMP level was also not elevated by apigcnin unless tb.c concentration of 370/z M was used (Table i).
Effects of apigeni, on NE-imhwed inositol monophosphate formation Exposure of rat aorta to 3/.tM NE for 15 ,nin in the presence of 1(I mM LiC! clcvatcd inositol monophosphate levels. Prctrcatmen: with apigcnin (140/.tM) l\~r 5 min did not inhibit the inositol monophosphatc tt~rmation caused by NE (Table I!). Effects of apigenin on 4~Ca2 ~ inJlltr bzdm'cd by NE or high K ÷ in the presence of NE (3 /zML the ~ ( ' a 2" influx was increased 41.8 + 1.9% over that in the control rings. This increase of "~SCa-'~ influx was inhibited by apigenin (35 to 140 # M ) in a concentration dependent manner (Fig. 5A). A similar inhibitory, effect on high K~'-induced 4SCa2" influx was also shown in Fig. 5B. High K ÷ (60 mM) caused a 51.4 +_ 3.(Jeff, increase of 45Ca2+ influx over that in the control rings, and this increase was also inhibited by apigcnin (35 to 140 txM) in a concentration dependent manner. Discussion
Vascular endothelium plays an important role in controlling the vascular tone via secretion cff both relaxant and contractile factors [17.18]. In response to a variety of neurohumoral and physical stimuli, endothelial cells release endothelium-dcpendent wlsodilators such as endothelium-dcrived relaxing factor (EDRF) and prostacyclin (PGI_,). The relaxing action of apigenin persisted in denuded or intact aorta in the presence of indomethacin (21)/.tM) or methylene bluc (50/zM). Indomethacin was known to block the generation of PGI.,, while methylene blue was reported to inhibit the E D R F activation of guanylatc cyclase [191~ Thus, the vasorelaxation caused by apigcnin was independent of the endothelium and was not mediated by either E D R F or PGI ~. Cyclic nucleotides are very important for relaxing vascular smooth muscle, cGMP causes Ca 2 + extrussion or sequestration [20,21] and inhibits Ca :~ uptake [22], contractile elements [23] or receptor-linked phosph0inositides breakdown [24,25]. cAMP can dilate vascular smooth muscle either by phosphorylation or" myosin light chain kinase [26,27], or increasing calcium uptake by the sarcoplasmic reticulum [28], or acting in other ways to reduce free cytosolic calcium [29]. It has been reported that flavonoids and biflavonoids are potent inhibitors of cyclic nucleotide phosphodiesterase [I,2], and may play roles in inhibition of platelct aggregation
and secretion [3-5]. Neither cAMP nor cGMP levels wcrc increased by apigenin at concentrations causing significant relaxation. This implies that the relaxing ,'ff,~ct of upigcnin is not duc to the formation of cyclic nuclcotidcs. Apigcnin inhibited the phasic contraction caused by NE. It has been reported that activation of o¢~-adrenergic receptors by NE stimulates phosphoinositides turnover to increase the concentration of inositot trisphosphatc and subsequently release the cellular ('a:" to mediate the phasic contraction [311,31]. However, NE-induccd inositol monophosphatc formation was nt~t inhibited by apigenin. This rules out the possibility that apigcnin may inhibit the NE-induced phasic contraction through inhibition of NE-induccd phosphoinositides hydrolysis. Apigenin also had no effect on caffeine-induced phasic contraction. It is now generally accepted that caffeine can release intracellular Ca: ~ in vascular smooth muscle and that there is an twcrlap between the NE- and caffeine-sensitive Ca 2~ stores [32-34]. Thcretorc, this result may imply that apigenin does not inhibit the release of intracellular Ca 2" from caffeine-sensitive as wctl as NE-scnsitive Ca 2 ~ stores. However. the action mechanism(s) of apigenin on the inhibition of NE-induced phasic contraction is not clear now, it may act on the site(s) after Ca 2+ was released. it is well known that high K'-induced contraction in smooth muscle is mediated by an increase in Ca-'* influx through voltage-gated Ca-"" channels [35]. Sincc apigenin inhibited Ca2%depcndent contraction and t~Ca2 ~ influx in high K ~ medium, it maybe a blockcr of voltage-gated Ca 2 ~ channel. The maintenance of tension (tonic contraction) in response to NE results primary from Ca -'~ entry through receptor-operated Ca-" channels with little requirement for Ca-' + entry through voltage-gated Ca-'" channel [36.37]. The NE-induced tonic contraction and "~SCa2 ~ influx were both inhibited by apigenin (Figs. ! and 5A). Furthermore, apigenin still relaxed the NEinduced tonic tension in a concentration dependent manner, in the presence of nifedipine (2 /zM, Fig. 3), which completely blocked high K + (80 mM)-induced contraction, in general, the membrane potential of vascular smooth muscle cell depolarized by NE was not more than 20 mV [38-40], and the increase of extracellular K ~ from 4.7 to 60 mM depolarized the vascular membrane potential about 60 mV [41] and about thc same value calculated as from Nernst equation assuming the intracellular K ~ is t50 mM. Thus, this indicates that apigcnin maybe a blocker of receptor-operated Ca 2 " influx. Rat aorta is different to rabbit aorta in some of its properties. In rabbit aorta, the voltage-gated Ca -'* channel and receptor-operated Ca z+ channel are independent. The former is activated by membrane depolarization and is more selectively blocked by organic
74 C a z+ c h a n n e l b l o c k e r s such as v e r a p a m i l , n i f e d i p i n e and diltiazem, w h e r e a s the latter is a c t i v a t e d by t h e b i n d i n g o f agonist to their r e c e p t o r s and is m o r e selectively b l o c k e d by s o d i u m n i t r o p r u s s i d e [22,42]. H o w -
ever, these two types of Ca z+ channels are functionally not completely separated in rat aorta [35,36], and voltage-gated Ca -'+ channel blocker (e.g., nitrendipine, nifedipine and verapamil) usually inhibited NE-induced contraction completely in higher concentrations [43]. Thus, in rat aorta, both of voltage-gated and r e c e p t o r - o p e r a t e d Ca 2+ channels are not totally separated and inhibited by apigenin. If apigenin is a selective inhibitor of receptor or voltage operated Ca 2+ channel in rabbit aorta needs further experiments to find out. It is concluded that the inhibitory effect of apigenin on thc c o n t r a c t i l e r e s p o n s e c a u s e d by high K + a n d N E in rat t h o r a c i c a o r t a is mainly d u e to inhibition o f Ca 2+
influx through both voltage- and receptor-operated Ca 2+ channels.
Acknowledgements T h i s w o r k was s u p p o r t e d by r e s e a r c h g r a n t s o f t h e N a t i o n a l S c i e n c e C o u n c i l of the R e p u b l i c o f C h i n a
(NSC79-0412-B002-26) and the National Research Institute of Chinese Medicine.
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