role of protein kinase c-mediated pathway in the pathogenesis of

2425

Role of Protein Kinase C-Mediated Pathway inthe Pathogenesis of Coronary Artery Spasm in

a Swine Model

Akira Ito, MD; Hiroaki Shimokawa, MD; Ryuichi Nakaike, MD; Tohru Fukai, MD;Makoto Sakata, MT; Tsuneo Takayanagi, MT; Kensuke Egashira, MD; Akira Takeshita, MD

Background The intracellular mechanism of coronary ar-tery spasm is still unknown. The pathway mediated by proteinkinase C (PKC) is an important intracellular process of variouscellular responses, including vascular smooth muscle contrac-tion. Thus, we examined the role of the PKC-mediated path-way in the pathogenesis of coronary artery spasm in our in vivoswine model.Methods and Results Seven Gottingen miniature pigs under-

went coronary balloon injury and x-ray irradiation to induceatherosclerotic lesion. After 6 to 18 months, intracoronaryserotonin (3 ,ug/kg) or histamine (3 ,ug/kg) repeatedly inducedcoronary artery spasm at the atherosclerotic site. At the spasticsite, intracoronary administration of phorbol-12,13-dibutyrate(PDBu) (10-9 mol/kg), a PKC-activating phorbol ester, alsoinduced coronary artery spasm, which was completely blockedby pretreatment with intracoronary staurosporine (10 ,g/kg), aPKC inhibitor. Intracoronary administration of an inactivephorbol ester, phorbol-12,13-didecanoate (10-9 mol/kg), didnot induce coronary vasoconstriction. Coronary artery spasm

C oronary artery spasm plays an important role inthe pathogenesis of a wide variety of ischemicheart diseases, not only in variant angina but

also in unstable angina, myocardial infarction, ventric-ular arrhythmias, and sudden death.1-4 However, thepathogenesis of coronary spasm is still unknown, andthe elucidation of its mechanism remains an importantclinical issue. We have developed a swine model ofcoronary spasm5-7 and revealed several pathogeneticaspects of the spasm. In our swine model, smoothmuscle contraction to autacoids (serotonin and hista-mine) is augmented, endothelium-dependent relax-ations to the autacoids are reduced,8'9 and calciumsensitivity of the contractile proteins in smooth muscleper se is not augmented at the spastic site.10 Thus, thekey mechanism(s) for smooth muscle hyperconstrictionappears to exist in the signal transduction pathway at alevel between receptors and the contractile proteins.The pathway mediated by protein kinase C (PKC) is

an important intracellular process of various cellular

Received March 18, 1994; revision accepted July 26, 1994.From the Research Institute of Angiocardiology and Cardiovas-

cular Clinic, Kyushu University School of Medicine, Fukuoka,Japan.

Correspondence to Hiroaki Shimokawa, MD, Research Insti-tute of Angiocardiology and Cardiovascular Clinic, Kyushu Uni-versity School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka812, Japan.

C 1994 American Heart Association, Inc.

induced by the autacoids was significantly augmented by pre-treatment with intracoronary PDBu and partially inhibited bystaurosporine. Intracoronary administration of Bay K 8644 (10,ug/kg), a dihydropyridine-sensitive L-type calcium channelagonist, also induced coronary artery spasm at the spastic site,which was significantly inhibited by pretreatment with intracor-onary staurosporine or nifedipine (0.1 mg/kg).

Conclusions These results suggest (1) the PKC-mediatedpathway is importantly involved in the pathogenesis of coro-nary artery spasm, (2) activation of the PKC-mediated path-way partially accounts for serotonin- and histamine-inducedcoronary artery spasm, and (3) at the spastic site, calciuminflux through dihydropyridine-sensitive L-type calcium chan-nel and/or calcium sensitivity of the contractile proteins maybe augmented by the PKC-mediated pathway. (Circulation.1994;90:2425-2431.)Key Words * calcium channels * atherosclerosis .

phorbol ester * vasoconstriction

responses," including vascular smooth muscle contrac-tion.12 Previous in vitro experiments have demonstratedthat phorbol-12,13-dibutyrate (PDBu), a PKC-activat-ing phorbol ester, induces sustained vasocontraction,13augments the sensitivity of contractile proteins to cal-cium ions (Ca2), suppresses the release and the actionsof endothelium-derived relaxing factor (EDRF),14"15and stimulates the release of endothelium-derived con-tracting factor (EDCF).16 However, little is knownabout the role of the PKC-mediated pathway in thepathogenesis of coronary artery spasm. Thus, the pres-ent study was designed to examine the role of thePKC-mediated pathway in the pathogenesis of coronaryartery spasm in our in vivo swine model.

MethodsAnimal Preparation

Seven male Gottingen miniature pigs, 2 to 4 months old andweighing 20 to 30 kg, were sedated with an intramuscularadministration of ketamine hydrochloride (12.5 mg/kg) andanesthetized with an intravenous administration of sodiumpentobarbital (25 mg/kg). The animals were then intubatedand ventilated with room air, and oxygen was supplemented bya positive-pressure respirator (Shinano Inc). Under asepticconditions, a preshaped Kifa catheter was inserted from thecarotid artery into the orifice of the left coronary artery underthe guidance of the fluoroscopy in a C-arm x-ray system(Toshiba). After pretreatment with heparin (3000 U IV),endothelial denudation about 3 cm long was performed on theleft anterior descending coronary artery (LAD) using a bal-

by guest on February 2, 2018http://circ.ahajournals.org/

Dow

nloaded from

http://circ.ahajournals.org/

2426 Circulation Vol 90, No 5 November 1994

loon catheter (2F Fogarty embolectomy catheter, EdwardsLaboratories).5-7 After recovery, they were fed 2% cholesteroldiet for 3 months. The denuded site was then selectivelyirradiated with x-rays (1500 rad) twice, 1 and 2 months afterthe coronary denudation.17 Animals were carefully treatedaccording to the guidelines of the American PhysiologicalSociety.

Coronary AngiographySix to 18 months after coronary denudation, the animals

were again anesthetized and ventilated as described above,and selective coronary arteriography was performed. A pre-shaped Judkins catheter was inserted into the right or leftfemoral artery, and coronary arteriography in a left anterioroblique view was performed. ECGs (leads 1, 11, III, V,, and V6)and blood pressure were continuously recorded during theexperiments. The angiographic study was performed twice atan interval of 1 to 2 weeks to examine the following twoprotocols.

Protocol 1Coronary artery vasomotion in response to intracoronary

administration of various vasoconstrictors was examined, in-cluding serotonin, histamine, phorbol-12,13-didecanoate(PDD), an inactive phorbol ester, and PDBu. Serotonin- andhistamine-induced coronary vasoconstriction was also exam-ined before and after intracoronary administration of PDBu orstaurosporine, a PKC inhibitor. In three pigs, coronary arteryvasomotion to prostaglandin F2a (PGFZa) was also examined.Coronary arteriography was performed 2 minutes after intra-coronary administration of serotonin (1 and 3 ,.g/kg), 1minute after that of histamine (1 and 3 ,ug/kg), and 5 minutesafter that of PGF2a (50 gg/kg), when hemodynamic variablesreturned to the control level. To examine coronary arteryvasomotion to phorbol esters, coronary arteriography wasrepeated at an interval of 5 to 10 minutes for 60 minutes afterintracoronary administration of PDBu (10- 0, 3x10-9, and10` mol/kg) and for 20 minutes after that of PDD (10`mol/kg). These doses of PDBu were determined in the pre-liminary study so that the estimated maximum concentrationof PDBu (10`9mol/kg) was 10-7 mol/L when the phorbol esterwas injected into the left coronary arteries for 3 minutes. Thesame dose (10` mol/kg) was chosen for PDD. The differentdurations of the coronary arteriographic study for PDBu (60minutes) and PDD (20 minutes) were determined based onthe preliminary findings that the vasoconstrictor effect ofPDBu (10` mol/kg) lasts for 60 minutes and that PDD (10`mol/kg) causes no coronary vasoconstriction. We also con-firmed in the preliminary study that the vasoconstrictor effectof PDBu was reproducible. Care was taken so that the injectedvolume of PDD and PDBu (dissolved in physiological saltsolution containing less than 5% dimethyl sulfoxide) was 6 mLand the injection time was 3 minutes. We confirmed in thepreliminary study that the dimethyl sulfoxide solution alonedid not affect the coronary diameter. Intracoronary staurospo-rine (10 ,ug/kg) was given 5 minutes before intracoronaryadministration of PDBu at 10` mol/kg, and coronary arteri-ography was repeated after that. To examine the effect ofactivation of the PKC-mediated pathway on serotonin- andhistamine-induced coronary vasoconstriction, coronary arteri-ography after intracoronary administration of those autacoidswas repeated 5 minutes after staurosporine (10 jug/kg) andmore than 60 minutes after PDBu (10-9 mol/kg) when thecoronary diameters almost returned to the control level. Atthe end of the experiments, coronary arteriography was re-peated after intracoronary administration of nitroglycerin (10gg/kg).

Protocol 2Coronary artery vasomotion was examined in response to

intracoronary administration of Bay K 8644, a dihydropyri-

dine-sensitive L-type Ca' channel agonist,'8 before and afterintracoronary administration of staurosporine and nifedi-pine.19 Coronary arteriography was performed 2 minutes afterintracoronary administration of Bay K 8644 (1, 3, and 10,g/kg). Intracoronary staurosporine (10 gg/kg) or nifedipine(0.1 mg/kg) was given 5 minutes before intracoronary admin-istration of Bay K 8644 (10 ag/kg).

Coronary Diameter MeasurementCineangiograms at end diastole were chosen and printed,

and the coronary luminal diameters were measured withcalipers. With this technique, excellent correlations betweenrepeated measurements (r=.99) and between different observ-ers (r=.98) were confirmed in the range of the coronarydiameter from 0.98 to 5.58 mm. The degree of constrictiveresponse was expressed as the percent decrease in the luminaldiameter from the control level. The coronary diameter wasmeasured at the segment of the LAD where a focal excessiveconstriction evoked with drugs was maximal and at the seg-ment of the left circumflex coronary artery (LCx) where thediameter was similar to that of the spastic segment of the LADunder control conditions.

Histological ExaminationAfter the angiographic experiments, hearts were removed

and the left coronary arteries were perfused by a constant-pressure perfusion system (120 cm H20) with saline (500mL) and subsequently with 6% formaldehyde (1000 mL).After the fixation, both the LAD and the LCx were cuttransversely into segments at a 5-mm interval along the maintrunk with small surrounded tissues. These segments werestained with hematoxylin-eosin and van Gieson's elasticstaining for photomicroscopy.

DrugsThe following drugs were used: 5-hydroxytryptamine (sero-

tonin), histamine, PGF2a,, PDD, PDBu, staurosporine (SigmaChemical Co), Bay K 8644 (Wako Junyaku Co), and nifedipine(Bayer Pharmaceutical Co). PDD, PDBu, and staurosporinewere prepared as stock solutions in dimethyl sulfoxide. Dilu-tion was made with a physiological salt solution. Nifedipinewas prepared by diluting the content of a nifedipine capsule(10 mg) in 10 mL of physiological salt solution.

Statistical AnalysisThe results were expressed as mean+SEM. Multiple com-

parisons were made by a two-way ANOVA followed by a posthoc test, and unpaired data were analyzed by Student's t test.A value of P<.05 was considered statistically significant.

ResultsAt the angiographic study, pigs weighed 35 to 47 kg.

Intracoronary serotonin (3 ag/kg) or histamine (3 gg/kg) repeatedly induced coronary spasm at the previ-ously denuded and x-irradiated LAD with ischemicECG changes (Fig 1). In contrast, PGF2, (50 ,g/kg)induced comparable vasoconstriction at the spasticLAD (28.2±7.9%, n=3) and at the control LCx(29.4±6.4%, n=3). At those spastic sites, PDBu (10`mol/kg) also induced coronary spasm with ischemicECG changes (Fig 1). The time course of phorbolester-induced coronary vasoconstriction is shown in Fig2. At the spastic LAD, PDBu (10` mol/kg) inducedmarked coronary vasoconstriction that peaked at 20minutes. On the other hand, at the control LCx, PDBuinduced only mild coronary vasoconstriction. ThePDBu-induced spasm at the LAD and the mild vaso-constriction at the LCx was completely blocked bypretreatment with intracoronary staurosporine (10 jug/


Dow

nloaded from


Ito et al PKC Pathway and Coronary Spasm 2427

A~~~~~~~~~~~~~~~~~~~~~~~~~~4 9

Xi\; V S

FIG 1. Coronary angiograms of the left coronary artery before (A) and after intracoronary administration of serotonin (3 jig/kg) (B),histamine (3 jig/kg) (C), and PDBu (10 9 mol/kg) (0). Arrows in B, C, and D indicate the spastic site of the previously denuded andx-irradiated site of the left anterior descending coronary artery. Isohemic EGG changes occurred with those spasms.

kg) (Fig 2). PDD (109 mol/kg) did not induce coronaryvasoconstriction at either site. PDBu-induced coronaryvasoconstriction was dose dependent and was signifi-cantly greater at the spastic LAD than at the controlLCx (Fig 3). Intracoronary administration of nitroglyc-erin (10 ,ug/kg) caused coronary vasodilatation at thespastic LAD (27.6±11.7%) and at the control LCx(7.8+3.6%, n=6, NS). Intracoronary administration ofstaurosporine (10 gg/kg) also caused coronary vasodi-latation at the spastic LAD (14.3+4.7%) and at thecontrol LCx (9.2±5.5%, n-5, NS).At the spastic site, serotonin- and histamine-induced

coronary spasm was significantly augmented by pre-treatment with intracoronary PDBu (10` mol/kg) (Figs

4 and 5), whereas it was partially inhibited by pretreat-ment with intracoronary staurosporine (10 jig/kg) (Figs4 and 5). However, even after the treatment withstaurosporine, vasoconstricting responses to the auta-coids were still greater at the spastic LAD than at thecontrol LCx. In contrast, at the control LCx, thisPDBu-induced augmentation of coronary vasoconstric-tion was not observed in response to either serotonin orhistamine, although the inhibition by staurosporine wasnoted in response to serotonin (Figs 4 and 5).Bay K 8644 (10 jgg/kg) also induced coronary spasm

at the spastic LAD (Fig 6). Bay K 8644-induced coro-nary vasoconstriction was dose dependent and signifi-cantly inhibited by pretreatment with intracoronary

80

60 [

40~

20

0

LCx (control)0 PDBu n=7

- PDD n=60--- after Staurosporine n=5

-,1 - _

>00~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

0 1 0 20 30 40 50 60

FIG 2. Line plots: Time course of PDBu-inducedcoronary vasoconstriction at the spastic left ante-rior descending coronary artery (LAD) and thecontrol left circumflex coronary artery (LCx). At thespastic LAD (left), PDBu (10-9 mol/kg) inducedmarked coronary vasoconstriction (open circles),which was completely blocked by pretreatmentwith intracoronary staurosporine (10 jig/kg)(closed circles). At the control LCx (right), PDBu(10-99mol/kg) induced only miid coronary vasocon-striction. PDD (10-1 mol/kg), an inactive phorbol

70 ester, did not induce any coronary vasoconstrictionat both sites (open square).

100

C0)._1

z

4

CD

0

z

eE_J

0 10 20 30 40 50 60 70

TIM E(min)

-20'

T ME(m in)


Dow

nloaded from



100 - * LAD (spastic)0 LCx (control)

= n=6j 80 p<o.05 vs LCxXh *- p<0.01 vs LCxZ 60

E 4o

~20

1 0 9.5 9

PDBu (-Log mol/kg)

FIG 3. Plot shows dose-response relation of percent luminalnarrowing in response to PDBu. PDBu-induced coronary vaso-constriction was dose dependent and was significantly greater atthe spastic left anterior descending coronary artery (LAD)(closed circles) than at the control left circumflex coronary artery(LCx) (open circles).

staurosporine (10 gtg/kg) or nifedipine (0.1 mg/kg)(P<.01) (Fig 6).

Histologically, intimal and medial thickening, whichwas compatible with atherosclerotic changes, was notedat the spastic LAD but not at the control LCx (Fig 7).Full lining of regenerated endothelial cells at the spasticsite was confirmed by electron microscopy, as we re-ported in the previous study.20

DiscussionThe major findings of this study were (1) PDBu, a

PKC-activating phorbol ester, induced coronary spasmat the atherosclerotic site, where serotonin and hista-mine also induced the spasm, (2) coronary spasminduced by serotonin and histamine was attenuated bypretreatment with staurosporine, a PKC inhibitor, butvasoconstricting responses to the autacoids were stillgreater at the spastic than at the control site even afterpretreatment with staurosporine at a concentration thatabolished the PDBu-induced coronary spasm, and (3)Bay K 8644, a dihydropyridine-sensitive L-type Ca2+channel agonist, also induced coronary spasm at thespastic site, which was inhibited by pretreatment withstaurosporine or nifedipine. To our knowledge, this isthe first report that demonstrated the important role ofPKC-mediated pathway in the pathogenesis of coronaryartery spasm in vivo.

PDBu-Induced Coronary Artery SpasmThe previous in vitro experiments have shown that

PKC-activating phorbol esters induce sustained coro-nary contraction.13 However, the mechanism of phorbol

ester-induced vasocontraction is unknown. Fish et a121reported that 12-O-tetradecanoyl phorbol-13-acetate,an active phorbol ester, increased the dihydropyridine-sensitive calcium conductance in the A7r5 rat aorticsmooth muscle cell line. Other investigators have re-ported that phorbol ester-induced vasocontraction isnot accompanied by a change in intracellular calciumlevel,2223 which implies that calcium sensitivity of thecontractile proteins may be augmented by activatingPKC with phorbol esters. Furthermore, PDBu inhibitsendothelium-dependent relaxations in canine coronaryarteries by inhibiting both synthesis/release and actionsof EDRF.14,15 PDBu also stimulates the release ofEDCF from canine femoral arteries.16 Despite these invitro findings, little is known about the in vivo effects ofphorbol esters on vascular responses, especially in rela-tion to the pathogenesis of coronary spasm.The present study demonstrates that PDBu, a PKC-

activating phorbol ester, induced vasoconstriction inporcine coronary arteries in vivo and that the vasocon-striction was significantly greater at the spastic than atthe control site in our swine model. The marked coro-nary vasoconstriction induced by PDBu (10` mol/kg) atthe spastic site was accompanied by ischemic ECGchanges. Thus, PDBu caused coronary spasm withmyocardial ischemia, as did serotonin and histamine.Interestingly, however, PDBu-induced spasm lasted fora longer duration (response peaked at 20 minutes andwas sustained for 50 to 60 minutes) than that induced byserotonin (response peaked at 2 to 3 minutes and wassustained for 5 to 7 minutes) or histamine (responsepeaked at 1 minute and was sustained for 3 to 4minutes). These results are consistent with the in vitrofinding that PDBu induces slowly developing contrac-tion of isolated porcine coronary artery.24 PDBu-in-duced coronary spasm was completely blocked by pre-treatment with intracoronary staurosporine. An inactivephorbol ester, PDD, did not induce coronary vasocon-striction. These results suggest that the PKC-mediatedpathway in vascular smooth muscle is importantly in-volved in the pathogenesis of coronary spasm in ourswine model. We also confirmed in the subsequent invitro studies that responsiveness of vascular smoothmuscle to PDBu was greater in vessels taken from thespastic site than in those from the control site (unpub-lished observations).

In the present study, PDBu was administered intra-coronarily at an estimated concentration of 10`7 mol/Lonly for 3 minutes. The vasoconstrictor effect of PDBuwas reproducible. These results suggest that the intra-

LAD (spastic)0 Control n=7

after PDBu n=7- after Staurosporine n=4

* p<0.05 vs Control |?.I3

*ca

.E

J

3

Serotonin (1tg/kg)

LCx (control)0 Control n=7* after PDBu n=7

after Staurosporine n=4pp<0.05 vs Control

1 3

Serotonin (Rg/kg)

FIG 4. Line plots: Serotonin-induced coronary vaso-constriction at the spastic left anterior descendingcoronary artery (LAD) (left) and the control left circum-flex coronary artery (LCx) (right). Serotonin-inducedspasm (open circles) was significantly augmented bypretreatment with intracoronary PDBu (10-9 mol/kg)(closed circle), whereas this spasm was partially inhib-ited by pretreatment with intracoronary staurosporine(10 ,g/kg) (closed square) (left). Note that this aug-mentation was not observed at the control site, al-though the inhibition by staurosporine was observedin response to serotonin (right).

CD

.30

z

.EE

mJ_it

100

80

60

40

20


Dow

nloaded from



LAD (spastic)

0 Control n=70 after PDBu n=7* aflter Staurosporine n=4

* p<0.05 vs Control

1X*1 3

100

C

3:

0!m

z1aZC

E.i

80

60

40

20

0

Histamine ([tgIkg)

LCx (control)

0 Control n=7* after PDBu n=7

-* after Staurosporine n=4

1 3

Histamine ([tgIkg)

FIG 5. Line plots: Histamine-induced coronary vaso-constriction at the spastic left anterior descendingcoronary artery (LAD) (left) and the control left circum-flex coronary artery (LCx) (right). Histamine-inducedspasm (open circles) was significantly augmented bypretreatment with intracoronary PDBu (10-9 mol/kg)(closed circle at 1 ,tg/kg histamine), whereas thisspasm was partially inhibited by pretreatment withintracoronary staurosporine (10 ,ug/kg) (closedsquare) (left). Note that neither augmentation by PDBunor inhibition by staurosporine was observed at thecontrol LCx (right).

coronary administration of PDBu did not cause a

sustained, intense activation of PKC that might lead toa downregulation of the enzyme.25

IfPKC is tonically activated at the atherosclerotic andspastic sites under basal conditions, a smaller responseto PDBu (because of the reduced availability of PKC)and a greater vasodilation to staurosporine would beexpected at the spastic site, which was not the case inthe present study. PKC-mediated sustained vasocon-striction may be operative in the cerebral vasospasmafter subarachnoid hemorrhage, where PKC is tonicallyactivated under basal conditions and the spontaneous,diffuse vasospasm lasts for days to weeks.26 This type ofvasospasm is in contrast to the coronary spasm in ourmodel, where PKC-mediated responses are augmentedonly upon stimulation. Thus, we interpret our data asthat the mass of and/or the substrate for PKC may beincreased at the atherosclerotic and spastic sites andthat PKC-mediated responses are augmented only uponstimulation but not under basal conditions. Althoughthis interpretation should be examined in future studies,we have recently demonstrated in vitro that the PKC-mediated pathway is also importantly involved in thehypercontraction to serotonin of isolated vascularsmooth muscle from Watanabe heritable hyperlipid-emic rabbits.27

PKC-Mediated Pathway in Serotonin- andHistamine-Induced Coronary Artery Spasm

In the present study, serotonin- and histamine-in-duced coronary vasoconstriction was augmented bypretreatment with PDBu at the spastic but not at thecontrol site in vivo. Miller et a128 examined the role ofthe PKC-mediated pathway in regulating the contractileresponses of isolated coronary arterial smooth muscleto a variety of stimuli in both intact and skinnedcoronary artery strips. They demonstrated that 12-0-

i

0c-

z

-i

wEE

100

80

60

40

20

0'

LlAD (spastic)LCx (control)

n=4* p<O.05 vs LCx**p<0.01 vs LCx

1 3 10

Bay K 8644 ([tgIkg)

'E3

O Control n=4E3 after Stauros* after Nifedipi* p<0.05 vs C

60-

40-

20

0

-20LAD

tetradecanoyl phorbol-13-acetate produced a leftwardshift of the concentration-response relation of K'-,histamine-, and norepinephrine-induced contraction.They concluded that the PKC-mediated pathway regu-

lates contractile responses of coronary arterial smoothmuscle to a variety of stimuli at least in part byincreasing the sensitivity of the contractile proteins toCa2+.28 The results of the present study are consistentwith those previous findings.

However, in the present study, serotonin- and hista-mine-induced coronary spasm was only partially inhib-ited by pretreatment with staurosporine at a concentra-tion of 10 ,ug/kg, which abolished the PDBu-inducedvasoconstriction; hyperconstriction to those autacoidsstill existed even after the PKC inhibition by staurospo-rine. In our swine model, the blockers of serotonin(ketanserin) and histamine (diphenhydramine) com-pletely prevented the coronary spasm induced by sero-tonin and histamine, respectively, indicating that thesignals for the hyperconstriction enter into the vascularsmooth muscle exclusively through those receptors.56Thus, these results may suggest that pathways otherthan the PKC-mediated one located downstream fromthe autacoid receptors are also involved in the patho-genesis of the spasm induced by the autacoids. Theresults may also suggest that the concentration of stau-rosporine used in the present study was relatively selec-tive for PKC inhibition.

Bay K 8644-Induced Coronary Artery SpasmBay K 8644, a dihydropyridine-sensitive L-type Ca2'

channel agonist, also induced coronary spasm at thespastic site in our swine model. The finding that Bay K8644-induced spasm was significantly inhibited by pre-treatment with staurosporine suggests the importantrole of the PKC-mediated pathway in the Ca2' channelagonist-induced spasm. A previous report from our

FIG 6. Graphs illustrate Bay K 8644-induced coro-

sporine n=3 nary vasoconstriction. Left, Dose-response relation ofdne n=3 percent luminal narrowing to Bay K 8644. Bay K,ontrol 8644-induced coronary vasoconstriction was dose

dependent and was significantly greater at the spasticleft anterior descending coronary artery (LAD) (closedcircles) than at the control left circumflex coronary

i ~ artery (LCx) (open circles). Right, Bay K 8644 (101,g/kg)-induced spasm at the LAD (open bar) wassignificantly inhibited by pretreatment with intracoro-nary staurosporine (10 ,ug/kg) (hatched bar) or nifed-

LCx ipine (0.1 mg/kg) (closed bar). These inhibitions werealso observed at the control left circumflex coronaryartery.

100

80

60h

Cs

0

z

M

E3_i

40

20

r

0

100

80


Dow

nloaded from



FiG 7. Light micrographs of the spastic left anterior descendingcoronary artery (top) and the control left circumflex coronaryartery (bottom). Intimal and medial thickening, which was com-patible with atherosclerotic changes, was noted at the spastic leftanterior descending coronary artery but not at the control leftcircumflex coronary artery (magnification x40).

laboratory has shown that in our swine model there isno difference in the calcium-tension relation of thecontractile proteins in the skinned arterial strips be-tween the spastic and the control site, indicating thatcalcium sensitivity of the contractile proteins per se isnot augmented at the spastic site.10 Fish et a121 demon-strated that activation of PKC increases the dihydropyr-idine-sensitive Ca>2 conductance in a vascular smoothmuscle cell line. Thus, the results in the present studysuggest that Ca2+ influx through the dihydropyridine-sensitive L-type Ca2+ channel and/or calcium sensitivity

of the contractile proteins may be augmented by thePKC-mediated pathway.

Role of PKC-Mediated Pathway in thePathogenesis of Coronary SpasmAs discussed earlier, we speculate that the mass of

and/or the substrate for PKC may be increased at theatherosclerotic and spastic site. It is also possible thatthe signal transduction for vasoconstriction is aug-mented at a certain step of PKC-mediated pathway.Indeed, the role of the PKC-mediated pathway in thevascular smooth muscle hyperconstriction may be com-plex, and multiple sites might be involved in addition toPKC itself. Those include the dihydropyridine-sensitiveL-type Ca> channel, sarcolemmal receptor-operatedCa> channels and the sarcoplasmic reticulum (SR)inositol triphosphate (IP3)/Ca2+ release channel (mech-anisms allowing entry of Ca' into the cytosol uponactivation), and the sarcolemmal and SR Ca2 pumpsand the sarcolemmal Na+/Ca' exchanger (mechanismsremoving Ca' from the cytosol).29 Our data suggestthat among those possible mechanisms, at least theinteraction between the PKC-mediated pathway andthe dihydropyridine-sensitive L-type Ca>2 channel maybe augmented at the spastic site.Although it was beyond the scope of this study to

examine all of the possible mechanisms, another impor-tant clue was that PGF2a induced comparable coronaryvasoconstriction at the spastic and control sites. Thisfinding also has been repeatedly observed in our previ-ous studies.20 In our swine model, phenylephrine6 and athromboxane analogue30 also caused comparable coro-nary vasoconstriction at the spastic and control sites.These results may not only exclude the major contribu-tion of the geometric theory31 but also suggest thatcoronary hyperreactivity is developed selectively tosome agonists but not to others, probably due to thealtered intracellular signal transduction mechanisms.Recently, it has been reported that in the rat basilarartery PKC mediates vasoconstrictor responses to sero-tonin but not to prostaglandin F2a in vivo.32 Recently,also we have observed that the vasospastic responses ofporcine coronary arteries to serotonin and histaminebut not the vasoconstriction to prostaglandin F2a weresignificantly inhibited by staurosporine in ViVo.33 Incontrast, ryanodine, which inhibits the Ca2'-inducedCa> release from the SR store,34 significantly inhibitedthe vasoconstriction of porcine coronary arteries toprostaglandin F2, but not the vasospastic responses toserotonin or histamine in vivo.33 These results suggestthat the PKC-mediated pathway is important for thevasospastic responses induced by serotonin and hista-mine, whereas ryanodine-mediated calcium release maybe important for the vasoconstriction induced by pros-taglandin F,a. Further studies are needed to clarify themechanisms for the selective hyperreactivity at thespastic site.

Clinical ImplicationsAn intimate relation between coronary spasm and

atherosclerotic changes has been repeatedly confirmedin our swine model.5-9 Atherosclerosis is the inflamma-tory/proliferative process in which the PKC-mediatedpathway may play an important role.35 The presentstudy suggests that the PKC-mediated pathway is im-

.9t.


Dow

nloaded from



portantly involved in the pathogenesis of coronaryspasm in our swine model. If this is also the case forhuman coronary artery spasm, the present findings mayhave important clinical implications in understandingthe pathogenesis of the spasm and in developing newertherapeutic interventions to prevent and manage it.

AcknowledgmentsThis work was supported in part by grants from the Ministry

of Education, Science, and Culture (No. 02404045, No.04670540, and No. 05454274), Tokyo, Japan, the SandozFoundation for Gerontological Research, and the Japan Re-search Foundation for Clinical Pharmacology. The authorswish to thank Drs Takeo Itoh and Junji Nishimura, KyushuUniversity, and Dr Kouichi Saida, Bayer Pharmaceutical Co,for valuable comments on the manuscript and Chikako Towa-tari and Tomoko Takebe for technical assistance.

References1. Maseri A, Severi S, DeNes DM, L'Abbate A, Chierchia S, Marzilli

M, Ballestra AM, Parodi 0, Biagini A, Distante A. 'Variant'angina: one aspect of a continuous spectrum of vasospastic myo-cardial ischemia: pathogenetic mechanisms, estimated incidenceand clinical and coronary angiographic findings in 138 patients.AmJ Cardiol. 1978;42:1019-1035.

2. Hills LD, Braunwald E. Coronary artery spasm. N Engl J Med.1978;299:695 -702.

3. Shepherd JT, Vanhoutte PM. Spasm of the coronary arteries:causes and consequences (the scientist's viewpoint). Mayo ClinProc. 1985;60:33-46.

4. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesisof coronary artery disease and the acute coronary syndromes: part1. N Engl J Med. 1992;326:242-250.

5. Shimokawa H, Tomoike H, Nabeyama S, Yamamoto H, Araki H,Nakamura M, Ishii Y, Tanaka K. Coronary artery spasm inducedin atherosclerotic miniature swine. Science. 1983;221:560-562.

6. Shimokawa H, Tomoike H, Nabeyama S, Yamamoto H, Ishii Y,Tanaka K, Nakamura M. Coronary artery spasm induced in min-iature swine: angiographic evidence and relation to coronary ath-erosclerosis. Am Heart J. 1985;110:300-310.

7. Egashira K, Tomoike H, Yamamoto Y, Yamada A, Hayashi Y,Nakamura M. Histamine-induced coronary spasm in regions ofintimal thickening in miniature pigs: roles of serum cholesterol andspontaneous or induced intimal thickening. Circulation. 1986;74:826-837.

8. Yamamoto Y, Tomoike H, Egashira K, Kobayashi T, Kawasaki T,Nakamura M. Pathogenesis of coronary artery spasm in miniatureswine with regional intimal thickening after balloon denudation.Circ Res. 1987;60:113-121.

9. Yamamoto Y, Tomoike H, Egashira K, Nakamura M. Attenuationof endothelium-related relaxation and enhanced responsiveness ofvascular smooth muscle to histamine in spastic coronary arterialsegments from miniature pigs. Circ Res. 1987;61:772-778.

10. Satoh S, Tomoike H, Mitsuoka W, Egashira S, Tagawa H, Kuga T,Nakamura M. Smooth muscles from spastic coronary arterysegments show hypercontractility to histamine.Am J Physiol. 1990;259:H9-H13.

11. Nishizuka Y. The role of protein kinase C in cell surface signaltransduction and tumor promotion. Nature (Lond). 1984;308:693-698.

12. Rasmussen H, Takuwa Y, Park S. Protein kinase C in the regu-lation of smooth muscle contraction. FASEB J. 1987;1:177-185.

13. Castagna M, Yoshimi T, Kaibuchi K, Sano K, Kikklawa U,Nishizuka Y. Direct activation of calcium-activated phospholipid-dependent protein kinase by tumor-promoting phorbol esters.J Biol Chem. 1982;257:7847-7851.

14. Rubanyi GM, Desiderio D, Luisi A, Johns A, Sybertz EJ. Phorboldibutyrate inhibits release and action of endothelium-derivedrelaxing factor(s) in canine blood vessels. J Pharmacol Exp Ther.1989;249:853-863.

15. Flavahan NA, Shimokawa H, Vanhoutte PM. Inhibition of endo-thelium-dependent relaxations by phorbol myristate acetate incanine coronary arteries: role of a pertussis toxin-sensitiveG-protein. J Pharmacol Exp Ther. 1991;256:50-55.

16. Rubanyi GM, Luisi A, Johns A. Phorbol dibutyrate stimulates therelease of diffusible endothelium-derived vasoconstrictor factor(s)from canine femoral arteries. Circ Res. 1991;68:1527-1531.

17. Nagasawa K, Tomoike H, Hayashi Y, Yamada A, Yamamoto T,Nakamura M. Intramural hemorrhage and endothelial changes inatherosclerotic coronary artery after repetitive episodes of spasmin x-irradiated hypercholesterolemic pigs. Circ Res. 1989;65:272-282.

18. Kanmura Y, Itoh T, Kuriyama H. Agonist actions of Bay K 8644,a dihydropyridine derivative, on the voltage-dependent calciuminflux in smooth muscle cells of the rabbit mesenteric artery.J Pharmacol Exp Ther. 1984;231:717-723.

19. Schramm M, Towart R, Lamp B, Thomas G. Modulation ofcalcium ion influx by the 1,4-dihydropyridines nifedipine and BayK 8644. J Cardiovasc Pharmacol. 1985;7:493-496.

20. Fukai T, Egashira K, Hata H, Numaguchi K, Ohara Y, TakahashiT, Tomoike H, Takeshita A. Serotonin-induced coronary spasm ina swine model: a minor role of defective endothelium-derivedrelaxing factor. Circulation. 1993;88:1922-1930.

21. Fish RD, Sperti G, Colucci WS, Claphan DE. Phorbol esterincreases the dihydropyridine-sensitive calcium conductance in avascular smooth muscle cell line. Circ Res. 1988;62:1049-1054.

22. Itoh T, Kanmura Y, Kuriyama H, Sumimoto K. A phorbol esterhas dual actions on the mechanical response in the rabbit mes-enteric and porcine coronary arteries. J Physiol. 1986;375:515 -534.

23. Jiang MJ, Morgan KG. Intracellular calcium levels in phorbolester-induced contractions of vascular smooth muscle. Am JPhysioL 1987;253:H1365-H1371.

24. Mori T, Yanagisawa T, Taira N. Phorbol 12,13-dibutyrateincreases vascular tone but has a dual action on intracellularcalcium levels in porcine coronary arteries. Naunyn SchmiedebergsArch Pharmacol. 1990;341:251-255.

25. Chiba K, Kato N, Kuroki T. Down regulation of phorbol diesterreceptors by proteolytic degradation of protein kinase C in acultured cell line of fetal rat skin keratinocytes. J Biol Chem.1986;261:13013-13018.

26. Matsui T, Takuwa Y, Johshita H, Yamashita K, Asano T. Possiblerole of protein kinase C-dependent smooth muscle contraction inthe pathogenesis of chronic cerebral vasospasm. J Cereb Blood FlowMetab. 1991;11:143-149.

27. Tagawa H, Tomoike H, Mitsuoka W, Satoh S, Kuga T, ShimokawaH, Nakamura M, Takeshita A. Hyperreactivity of aortic smoothmuscle to serotonin is related to the presence of atheroma inWatanabe heritable hyperlipidaemic rabbits. Cardiovasc Res. 1993;27:2164-2169.

28. Miller JR, Hawkins DJ, Wells JN. Phorbol diester alters the con-tractile responses of porcine coronary artery. J Pharmacol ExpTher. 1986;239:38-42.

29. Andrea JE, Walsh MP. Protein kinase C of smooth muscle. Hyper-tension. 1992;20:585-595.

30. Shimokawa H, Tomoike H, Nabeyama S, Yamamoto H, NakamuraM. Histamine-induced spasm not significantly modulated by pros-tanoids in a swine model of coronary artery spasm. J Am CollCardioL 1985;6:321-327.

31. MacAlpin RN. Contribution of dynamic vascular wall thickeningof luminal narrowing during coronary arterial constriction.Circulation. 1980;61:296-301.

32. Murray MA, Faraci FM, Heistad DD. Role of protein kinase C inconstrictor responses of the rat basilar artery in vivo. J Physiol.1992;445:169-179.

33. Kadokami T, Shimokawa H, Ito A, Fukumoto Y, Takayanagi T,Egashira K. Activation of protein kinase C induces coronaryvasospasm at the interleukin-1f8-induced inflammatory/prolif-erative lesion in a swine model in vivo. Circulation. 1994;90[part 2].

34. Ito K, Takakura S, Sato K, Sutko JL. Ryanodine inhibits therelease of calcium from intracellular stores in guinea pig aorticsmooth muscle. Circ Res. 1986;58:730-734.

35. Uratsuji Y, DiCorleto PE. Growth-dependent subcellular redistri-bution of protein kinase C in cultured porcine aortic endothelialcells. J Cell PhysioL 1988;136:431-438.


Dow

nloaded from


TakeshitaA Ito, H Shimokawa, R Nakaike, T Fukai, M Sakata, T Takayanagi, K Egashira and A

in a swine model.Role of protein kinase C-mediated pathway in the pathogenesis of coronary artery spasm

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 1994 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation doi: 10.1161/01.CIR.90.5.2425

1994;90:2425-2431Circulation.

http://circ.ahajournals.org/content/90/5/2425World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://circ.ahajournals.org//subscriptions/

is online at: Circulation Information about subscribing to Subscriptions:

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

document. Permissions and Rights Question and Answer this process is available in the

click Request Permissions in the middle column of the Web page under Services. Further information aboutOffice. Once the online version of the published article for which permission is being requested is located,

can be obtained via RightsLink, a service of the Copyright Clearance Center, not the EditorialCirculationin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:


Dow

nloaded from

http://circ.ahajournals.org/content/90/5/2425

http://www.ahajournals.org/site/rights/

http://www.lww.com/reprints

http://circ.ahajournals.org//subscriptions/


role of protein kinase c-mediated pathway in the pathogenesis of

Documents