Mechanism of contractile responses to bradykinin in canine basilar arteries

?I&

study WBS WX%X~~KII

to i~~~~tig~it~ the rdc of cndctthclium’in

the modul:t!ion

of rascul;u- responses to bradykjnin

and to

clucidatc the rcccptor types and mechanism of action of hradykinin in isolated basilar artery. The results showed a contr~~iiic rcsponsc ((1 hradykinin in basilar artcry. This contractile rcsponsc to hradykinin was partially modulated by cndothclium in a dose-dcpendcnt antagonized

manner.

In addition.

Ph~‘]b~dykinin

(PA,

= 9.6 + 11.4) were

suggest that two ~p~rcn[Iy hradykinin (II)

“Ml

in basilar and TMB-K

artcry

was significantly EGTA

the n~~chanisn~ of the contractile

and BK,

greater

the blocking

than

those with

inhibited

(IO ’ M), but not by indomcthacin

that part of the contractile

on the BK,

Howcvcr.

distinct types of BK receptors

nifcdipinc. Ca’ ‘-free medium, indicating

both dcs-Arg“-[Lc$bradykinin

the rcsponscs to bradykinin.

and Ca’ ‘-free

and the apparent

dcs-Arg”-jLcu”]hrrtd~kinifi

may exist in basilar arteries.

(PA,

Furthermore,

= 7.X + 0.3).

significantly

rcduccd the hradykinin-induced

is depcndcnt

on cxtraccllular

in basilar artcry may involve increased inttaccllular

Endothclium:

1. Introduction Physiological and pathophysiological roles for hradykinin (BK), a vasoactivc nonopeptide, in the cardiovascular system have long been postulated (Haddy ct al., 1970). One of the chief physiological activities of BK is its ability to produce a fall in blood pressure when injected Lv.. However, its vas~~dilator activity is not consistently ohscrvcd in isolated blood vessels. on the spccics and anatomical

These

the contractile

results

response to

origin of the

of BK may elicit relaxation,

contraction.

Ca”.

In conclusion.

Ca”

rcccptor, ~~Jli~~w~d hy ~c~iv~ti(~n of the ph~)sph~~inositid~ ~tl~~v~y and r~c~pt(~r-m~di~l~d

blood vcsscl, applications

significantly

of [d-Arg”.Hyp~.Thi:‘.X.D-

by 2-nitro-4-carboxyphcnyl-N.N-diphcnylcarbamate (10 5 Ml. H-7 acid (IO ’ Ml. On the other hand.

mcdium/EGTA

rcsponscs to br~dykinin

affinity

(IO ’ M) or nordihydroquariarctic

response of basilar artcry lo bradykinin

Nradykinin:

Dcpcnding

and [d-Arg”,HypZ.Thi”~X.D-Phe7]bradykinin effect

lcvcls acting Ca”

channcf.

Basilar artery (canine)

blood VCSSCISstudied (Caldwcll et al., 1976: Ryan et al,. IY76;rl and (cl the release of other possible end~?genous mediatars from cndothelial cells (Furchgott. 1YXI; Cherry et al., 1982). Previous studies have dcmonstratcd vasodilator responses to BK in isolated rings of canine coronary, renal and mescnlcric artcrics. and of rabbit cerebral artery (Toda. 1977; Wahl et al., 1983). Also, BK has been shown to relax human cerebral artcry in vitro (Toda, 1977: Hardebo et al.. 19X5). Moreover, BK may have a significant effect on cerebral :r?d lsiuod-brain barrier function hemodynamics

contraction or no response at all. In addition, the action of BK on va$.cular preparati~~ns is known to

(Kamitani ct al., l%Sa. h). Our preliminary results showed a c~~tlt~-~ctilcresponses to BK in canine basilar

depend on scvcral factors including: (a) the BK rcccptor types present on the tissue, two of which have been

artery. This study was undertaken to elucidate the vascular response to BK of rings of basilar artcry from dogs. to

charactcrizcd recently (Regoli metabolic factors, in particular

and Barabd. IYXO~; Ih) catabolic enzymes such

as kininase II which has been suggested by Erdiis (1970) to be the same as ~lngi~)tensin converting cnzymc (ACE, pcptidyl dipeptidasc), that is present on the luminal

surface of vascular endothelial

cells of

invcstigatc which of the two reccpior types. BK, or BK ?. is involved in these responses and the role of end~~th~lial factors in canine basilar artery.

all 2.

Materialls and methods Mongrel

anesthetized

dog.; of cithor sex, weighing 7- IS kg. were with an i.p. injection of sodium pcntobar-

hit:tf (30 mg kg I I) and killed by exsanguination from the common carotid arteq. The bruins were rapidly rcmovcd and the basilar artery (0.6-0.8 mm outer di~~met~r) was isolated and placed in a physiofo~ical saft sdution (composition in mM: NaCf 118; KC1 4.7: NaHCO, 2% KH,POa 1.2 MgSf, 1.2% CaCf, 2.5 and gluccx;i\ 1I), bubbled with a mixture of 95% 0, and 5% CO,. Fat and loosely adhering connective tissue were removed. Rings were prepared by cutting the vascular segments transversely with fine-tipped scissors. Care was taken to minimize rubbing of the intimaf surface to prepare rings with intact, functional cndothefium. When necessary, endothefium was removed by rubbing the intimaf surface with a wooden stick. Each vascular ring was mounted in a 20-ml jacketed tissue bath ~ntaining a pbysiofogi~a~ sa!t solution maintained at 37°C and pH 7.3-7.4 by bubbling with a mixture of 95% 0, and 5% CO,. The resting tension was adjusted to 1 g. Mechanical activity was monitored isometrically with a Grass FTO3C force-displacement transducer (Quincy. MA, USA). Isometric force was measured and remrded with a Grass Model 7D pofygraph (Grass lnstrumcnt Company, Quincy, MA, USA). AI1 preparations were allowed to cquifibratc for 60-90 min in the bathing media before exposure to agonists. Each basilar arterial ring was tested for the presence or absence of endothefium by assessing the effects of 11)07 M acetyfcholine (AChI. The lack of a contractile response to IO-’ M ACh was taken as evidence for the absence of a functional endothclium (Usui et al., 198X Denuded and intact rings were also checked by scanning electron microscopy (Yen et al.. f%X). In order to avoid rapidly developing tachyphyfaxis to the contractife action of BK. increasing concentrations were

added separately and each preparation was always thoroughly washed out before it was exposed to a second conccntratlon of BK. Each ring was exposed to BK (3 x 10-“‘-lO-h MI for 10 min, followed by rinsing and a 30-min recovery period before the next concentration was added. With the exception of indomethacin pretreatment, 30 min prior to the agonists, all the other pretreatment drugs were added 10 min before. fn some experiments, contractile responses to BK were determined in a Ca’*-free solution. The Ca’+-free solution was prepared by omission of Ca’ + from the physiological salt solution and addition of EGTA at a concentration of 0.1 mM. pAI determination: in each experiment, agonist dose-response curves in the presence of BK, and/or BK2 receptor antagonist were related to the control dose-response curve, the maximum response of which was taken as 100%. In most experiments, three or four concentrations (IO-“, IO-‘, IO-’ and 10mh MI of antagonist were tested and sfopes of the resulting Schifd plots were used to assess competitive antagonism. The pA, values were calculated for each concentration of antagonist according to: pA, = -log ([antagonist]/ [dose-ratio - I]) (Taffarida et al., 1979). The results are expressed as the means + s.e. mean accompanied by the number of operations. BK, des-Arg’-BK t BK ,receptor agonist), des-Arg’?[LeuX]BK (BK, receptor antagonist) and d-Arg”‘[Hyp”, Thi5.X,d-Phe7]BK (BKI receptor antagonist) (Steranka et al., 1989; Bathon and Proud, 1991) were purchased from Peptide Co. (Osaka, Japan). 2-Nitro4_carboxyphenyl-N,N-diphenyfcarbamate (NCDC, phosphofipase C inhibitor; Wafanga et al., 1980; Nakaki et al., 19851,

a Basilar

Artery

b

2.0.

e-0 o-o

*

4-E -2

j/a

* ;.4--_i

*

*

_ -_O

0 9

8

7

8

-log CBKl (W Fig. 1. (at Typical responsr nhhtainrdwhen isnlatcd intact basilar artery WS exposed to hradykinin in increasing concentrations. Calibration bars are inserted. Conccnlralinns of bradykinin are shc~wnhctow Ihc Iraccs. (ht Concentration-responses CUIWS of hradykinin in intact artery (01 and mdnthetium-denuded artery (01. Mean values are shown with vertical lines indicating S.E.M.; number of vessels. 16. Statisticatty significant difference between intact and denuded using Student’s t-test is shown as * P < ft.05

‘h?

H-7 (protein kinase C inhibitor; Khalil and Van Breemen, 1988; Ratz, 19901, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxy benzoate hydrochloride (TMB-8) (Chiou and Malagodi, 19751, indomethacin (cyclooxygenase inhibitor), nordihydroquariaretic acid (NGDA, lipoxygenase inhibitor; Yen and Lee, 1987) and EGTA were purchased from Sigma Co. (St. Louis, MO, U.S.A.). Tetramethylpyrazine (Wu et al., 1989) was purchased from Aldrich Chem. Co. (Milwakee, WI, U.S.A.). The data are expressed as means + S.E.M. Significance was tested with Student’s I-test. Values of P < 0.05 were accepted as statistically significant.

3. Results 3.1. Role of endothelium in the concentration-responses cunr

to BK in basilar arteries

3.2. Distribution of types of BK receptor in basilar artery Figure 2 and table 1 show that the BK-induced contractile response in basilar arteries was almost completely abolished by the BK, receptor antagonist (5 X IO-’ M) but only partially blocked by the BK, recep-

ElKI-Ant B+K

I

Apparent affinirs

of BK, and BK,

receptor antagonist in canine

basilar artery. Values represent means f S.E.M.. numher of vessels= IO. Sl~pc of Schild plots is in parentheses. PA,

Basilar artery

BK,

BKI

7.14+0.17 ( I .0x * 0.02)

9.41 +o.OY (II.99 + 0.04)

tor antagonist (5 x lo-’ M). However, the BK, receptor agonist alone produced a small contractile response. Furthermore, table 1 shows that the apparent affinities for antagonists (pA2) to BK, and BK, were 7.14 f 0.17 and 9.41 f 0.09, respectively. 3.3. Effets of indomethacin and NDGA on the contractile response to BK in basilar arteries

Figure la and lb demonstrate that the contractile response to BK in intact basilar arteries was concentration-dependent. The response to BK in intact arteries was significantly greater than that in denuded basilar arteries. EDs,, values for BK in intact and denuded basilar artery were 6 X 10vq M and 9 X IO-” M. respectively. It thus appear that the contractile response to BK is partially endothelium-dependent.

BK

TABLE

BKa-Ant

In order to test whether the partial endothelium-dependent response to BK in basilar artery is mediated by the release of thromboxane (TX) AZ and/or leukotrienes from endothelium in basilar artery, basilar arteries were pretreated with NDGA or indomethacin. Figure 3 shows that indomethacin and NDGA did not affect the BK-induced contraction in endothelium-intact basilar artery. 3.4. Effects of NCDC, H-7. TMB8

and TMP on the BK-induced contraction in basilar artery

Figure 4 showed that both NCDC, a phospholipase C inhibitor, and H-7, a protein kinase C inhibitor, significantly inhibited the contractile responses to BK. Also, TMB-8 and TMP, two known inhibitors of Ca’+ release from sarcoplasmic reticulum (Wu et al.. 19891, were used to examine the contractile response to BK.

BK,

B+K

Fig. 2. Cnnlraction induced by hradykinin (3 x IO- ” M) alone (0) or in the presence of a BK, receptor antagonist (5 x IO -’ Mb ( l ), or BK1 receptor antagonist (5x 10~~’ M) (RI 1. and the Bii, receptor agonist alone (IO-’ MI (R ); BK, alone increased the tension. hul not significantly; vertical bars indicate S.E.M., number of vessels, Ih. St;ctislically significant differences between either hradykinin :done vs. BK, receptor antagonist and/or BK, receptor antagonisl are shown as * P < 0.05

Blc

Indo &

NDGA B+K

Fig. 3. Bradykinin (3x 10 s Ml-induced contraction in basilar artery alone (a), or in the presence of indomethacin (10 ’ Mb (D ). or NDGA (I()-” M) (N): vertical hars indicate S.E.M.: numb I>! vessels, 15.

4. Discussion

The present study demonstrated that BK produced cot~ccntration-dependent eontraction of the canine isolated basilar artery in vitro. This is consistent with the previous report by Toda (1977). However, the role of endothclium in modulating the vascular response to BK and the mechanism of the contractile responses to BK in basilar artery of the dog have so far not been studied. As shown in fig. lb, the coneentration-rcsponsc curve of BK in denuded basilar artery was attenuated as compared to that of intact artery, indicating that contractile response to BK in canine basilar artery was partially endothelium-dependent. The contra&e response to BK in basilar artery could be an~gonizcd by either the BK, or BK, receptor antagonist (fig. 2). Thus, the two types of BK receptors, BK, and BKz, may co-exist in basilar artery. However, the blocking effect of the BK, receptor antagonist on the BK response and pA, (the apparent affinity) of BK, receptor antagonist was significantly greater than that of the BK, rcccptor antagonist (fig. 2 and t?bic I). These results suggest that the contractile response to BK in basilar artery is more sensitive to block by a BK, antagonist than by a BK, antagonist. There is evidence that, in some arterial preparations, BK can cause the release of endogenoux proataglandins since indomcthacin Wane, 1971), a cyclooxygenasc inhibitor, blocked the relaxant effects of BK in rat aorta (Taylor and Morris, 1983). In contrast, contraction instead of relaxation in response to BK was found in canine basilar artery in the absence of precontraction (fig. la and b). This result was different from the report by Kim (Kim et al., 1989); this may be due to BK superfused through basilar artery then being acted on by rings coronary, in the bioassay. Converting enzyme, a degradation enzyme of BK, is known to exist in endothclial and smooth muscle cells. Therefore, the content of BK in perfusatc may be destroyed before it acts on the bioassay rings. Thus, BK-induced relaxation may be due to its stimulation of the release of cndothclium-derived relaxing factor (EDRF) from superfused basilar artery, which then acts on the bioassay rings. In addition, Kim et al. (19899) have reported that a contractile response to BK was found when BK was added directly to the bioassay rings. Moreover, the BK-induced response!: in denuded artery were signifiwntly attenuated rather than potentiatcd (fig. lb), implying that responses to BK were‘ mediated partially via the release of ~ndotl~cliu~~-derived contracting factor (EDCF) instead of EDRF. Arachidonic acid metabolifes, such as thromborunc 4, zitd/z $zukotricnes, are not involved in the resplnnse of basilar arteries to BK, as neither indomethacm, a cyc!ooxygcnasc inhibitor, nor NDGA, a lipoxygenase inhibitor, suppressed BK-induced cont~etio~ (fig. 3), Thub, It q-

a

g

LO

.rl

2 g 0 BK

NCDC

W7

B+

&

TM%i3

B+K

TKP B+K

Fig. 4. Contraction-induced by hrdykinin (3 x 10 ’ M) alone f LJ 1. in rhe presence of ph~~sp~~~Iip~~~ t inhibitor, NCIX (10 ’ h+) (Mt. protein kinase C inhibitor. Ii-7 I IO ’ Mt (m ). or calciumrelcasc inbihitur.TMB-8f 111~’Mbf rPf and TMP (5 x 10 ’ M) (m 1: v&cai tarp indicateSE-M.: number of vessels. 16. Statis~icslly significant difference from hradykinin alone is shown as * P < 0.015.

These two agents si~ni~cantly reduced B&induced vasoconstriction. However, the inhibitory effects of NCDC and H-7 on the responses to BK were significantly greater than those of TMB-8 and TMP. This indicates that the mechanism of the contraction in response to BK imoives the phosphoinositide pathway.

In order to test whether the cxtraccIluIar Ca”!’ was responsible for the contractile responses to BK, nifedipine. Ca’--free medium, EGTA and Ca”-free medium/EGTA were added to the tissue bath. Nifedipine, Ca”-free medium and EGTA significantly reduced the contractile responses to BK (fig. .‘,I.

Fig. S. C‘ontraclion-in&cud hy h:dykinm ;dlme (3 Y 10 ’ Ml ( n L in fhe prescnc; trf nifdipinc (II!’ ’ MI (EI). CG’-frCC medium (191). ECTA (!I) ’ MI (81) and Ca”-free mzdium/PGTA (8): WAXI bar> indic;tte S.E.M.: numhcr of VW.&. IO. * P < (t&i. st;ttislicully Ggnific:mt rfiffercnces comparedwith the br;l~ykinin altmc.

265

pears that arachidonic acid metabolites may not be the sole cause of the contractile response of canine basilar artery to BK. It has been consistently observed that the vascular responses to most of the neurotransmitters and hormones are mediated by the activation of the phosphoinositide pathway (Miche!!, 1975; Berridge and Irvine, 1984). Receptor-induced activation of vascular smooth muscle is associated with increased phospholipase C (PLC) activity, resulting in the production of the second messengers, inosito! triphosphate UP,) and diacylglycerol (DAG) (Horwitz, 1990). IP, has been shown to cause the release of Ca 2+ from intracellular stores (Streb et a!., 1984; Burgess et il., 1984; Joseph et a!., 1984). Figure 4 demonstrated that NCDC, a phosphotipase C inhibitor, and TMB-8 and/or TMP Wu et a!., 198% two known inhibitors of calcium re!ease from sarcoplasmic reticulum, significantly reduced the contractile response to BK in basilar artery. In addition, the activation of protein kinase C in vascular smooth muscle may evoke alterations in ion channel activity (Litten et a!., 1987). The present study explored the ability of H-7, an inhibitor of protein kinase C, to abolish vascular contraction in response to BK in intact canine basilar artery (fig. 4). This result confirmed the report of Khalil and Van Breemen (1988) and Laher and Bevan (19893 in studies with various cell lines. These results further support the possibility that protein kinase C may be involved in the contractile responpe to BK in b:isi!ar artcry. Eberhard and Holz (1988) have reported that cytosolic Ca’ ’ is regulated by two possible pathways. The first pathway is through the direct receptor-mediated activation of IP3 which releases Ca” from intraccllufar stores, thus increasing cytosolic Ca2+. The second pathway is entry of Ca2+ into cells through depslarization or ligand-gated channels. Figure 5 shows that nifedipine, Ca2 ‘-free medium, EGTA and Ca’+-free medium combined with EGTA significantly inhibited the contractile response to BK. This indicates that part of the BK-induced contraction in basilar artery was dependent on Ca2+ influx from the extracellular medium. In conclusion, the mechanism of the contractile response of canine basilar arteries to BK may involve botlr BK, and BK, receptors followed by a&&ion of phosphosinositide pathway and Ca2+ in!lux.

Acknowledgements We would like to thank Dr. David l3ohr for a helpful discussion. This work wus supported by a Grant (NSC-7Y-t1412-BOl6-111) from the N~iti(~iitll Science Council. Taipei, Taiwan, ROC.

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