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(Circulation. 1996;94:190-196.)
© 1996 American Heart Association, Inc.

Articles

Coronary Artery Spasm Does Not Depend on the Intracellular Calcium Store but Is Substantially Mediated by the Protein Kinase C–Mediated Pathway in a Swine Model With Interleukin-1ß In Vivo

Toshiaki Kadokami, MD; Hiroaki Shimokawa, MD; Yoshihiro Fukumoto, MD; Akira Ito, MD; Tsuneo Takayanagi, MS; Kensuke Egashira, MD; Akira Takeshita, MD

the Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University School of Medicine, Fukuoka, Japan.

Correspondence to Hiroaki Shimokawa, MD, Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan.

	Abstract

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Background The intracellular mechanism for coronary artery spasm is still unknown. Since the protein kinase C (PKC)–mediated pathway and Ca²⁺ release from sarcoplasmic reticulum (SR) are important intracellular mechanisms of vascular smooth muscle contraction, we examined the possible role of these two mechanisms in the pathogenesis of coronary spasm in our swine model in vivo.

Methods and Results In 25 pigs, interleukin-1ß (IL-1ß) was applied chronically to the coronary arteries from the adventitia to induce an inflammatory/proliferative lesion. Two weeks after the operation, either intracoronary serotonin or histamine repeatedly induced coronary spasm at the IL-1ß–treated site. At those spastic sites, phorbol-12,13-dibutyrate, a PKC-activating phorbol ester, also induced coronary spasm, which was blocked by pretreatment with the PKC inhibitors staurosporine and sphingosine. Serotonin- and histamine-induced coronary spasm was also significantly inhibited by pretreatment with staurosporine, sphingosine, or nifedipine (an L-type Ca²⁺ channel antagonist) but not by ryanodine (an inhibitor of Ca²⁺-induced Ca²⁺ release from SR) or thapsigargin (an inhibitor of Ca²⁺-ATPase of SR). Bay K 8644 (an L-type Ca²⁺ channel agonist) also induced coronary spasm at the IL-1ß–treated site, which was significantly inhibited by pretreatment with staurosporine, sphingosine, and nifedipine. In contrast, coronary vasoconstriction induced by prostaglandin F₂ was not affected by pretreatment with staurosporine or sphingosine but was significantly inhibited by pretreatment with ryanodine, thapsigargin, or nifedipine.

Conclusions These results suggest that (1) PKC activation largely accounts for the serotonin- and histamine-induced coronary spasm; (2) at the spastic site, the calcium influx through L-type Ca²⁺ channels may be augmented via the PKC-mediated pathway; and (3) the Ca²⁺ release from the SR into the cytosol may not play a primary role in coronary spasm.

Key Words: vasospasm • cytokines • protein kinase C • calcium channels • signal transduction

	Introduction

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Coronary artery spasm plays an important role in the pathogenesis of a wide variety of ischemic heart diseases, not only in variant angina but also in unstable angina, myocardial infarction, ventricular arrhythmias, and sudden death.^{1 2 3 4} Little is known about the intracellular mechanism of the spasm, however, and its elucidation remains an important clinical issue.

We previously developed a swine model of coronary spasm with an atherosclerotic coronary lesion induced by a combination of balloon endothelial denudation and high-cholesterol feeding.^{5 6 7} In this original model, coronary spasm was repeatedly provoked by autacoids (serotonin and histamine).^{5 6 7} We also recently developed a new swine model of coronary spasm, in which chronic perivascular inflammation induced by IL-1ß resulted in arteriosclerotic changes and vasospastic responses to the autacoids of the coronary arteries.^{8 9}

When agonists bind to the serotonergic and histaminergic receptors, phospholipase C is activated, leading to the formation of IP₃ and diacylglycerol by the hydrolysis of phosphatidylinositol 4,5-bis-phosphate.¹⁰ IP₃ then binds to an IP₃ receptor on the membrane of the SR to mobilize the stored calcium ions (Ca²⁺) from the SR into the cytosol. Diacylglycerol activates PKC, which causes vasoconstriction and augments the Ca²⁺ sensitivity of contractile proteins.¹⁰ Thus, both the intracellular Ca²⁺ store and the PKC-mediated pathway are able to contribute to the pathogenesis of coronary spasm, although the relative importance of the two mechanisms remains to be clarified. Indeed, we recently observed that the PKC-mediated pathway plays an important role in coronary spasm in our original model¹¹ ; however, the role of the intracellular Ca²⁺ store was not evaluated in that study. Thus, the present study was designed to examine whether or not those two intracellular mechanisms for smooth muscle constriction are altered at the spastic site in our new swine model in vivo.

	Methods

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Animal Preparation
Twenty-five male Yorkshire pigs weighing 25 to 30 kg were sedated with ketamine hydrochloride (12.5 mg/kg IM) and anesthetized with sodium pentobarbital (25 mg/kg IV). The animals were then intubated and ventilated with room air; oxygen was supplemented via a positive-pressure respirator (Shinano Inc). Under aseptic conditions, a left thoracotomy was performed, and the proximal segments of the left anterior descending and circumflex coronary arteries were carefully dissected. The dissected segments of the coronary arteries were gently wrapped with cotton mesh that had absorbed 0.05 mL of sepharose bead solution either with or without recombinant human IL-1ß 2.5 µg.^{8 9} The sites of the treatment with IL-1ß and control beads were randomized.

This experiment was reviewed by the Committee on Ethics in Animal Experiments of the Kyushu University School of Medicine and was carried out according to the Guidelines for Animal Experiments of the Kyushu University School of Medicine and the Law (No. 105) and Notification (No. 6) of the Japanese Government.

Preparation of IL-1ß Beads
IL-1ß beads were prepared as follows^{8 9} : 1 g sepharose microbeads (45 to 165 µm in diameter) was added to 50 mL of 1 mmol/L HCl solution and resuspended in 20 mL NaHCO₃/NaCl solution with 1 mg IL-1ß. The beads were allowed to bind with IL-1ß at room temperature for 1 hour and then at 4°C overnight. After centrifugation at 1200 rpm for 5 minutes, the supernatant was separated, and the concentration of the remaining IL-1ß in the supernatant was measured by an ELISA.¹² The IL-1ß–bound beads in the pellet were resuspended in 20 mL NaHCO₃/NaCl solution and centrifuged four times at 1200 rpm for 5 minutes. Then the IL-1ß–bound beads were resuspended with Tris-HCl buffer solution for 1 hour and finally washed and resuspended so that the concentration of IL-1ß was 50 µg/mL. All preparations were performed under sterile conditions.^{8 9}

Since in our bead preparation most of the IL-1ß molecules are bound inside the beads by a covalent bond at the amino residues of the proteins, 1.2% or less of the IL-1ß molecules are actually bound to the surface of the beads and biologically active. Thus, when 2.5 µg of IL-1ß that is bound to the beads is applied to the coronary artery, 30 ng of IL-1ß is biologically active.⁸

Experimental Protocols
Two weeks after the operation, we performed a coronary arteriographic study in which the coronary artery vasomotion in response to intracoronary administration of various vasoconstrictors was examined.

We examined coronary vasomotion in response to the intracoronary administration of serotonin 10 µg/kg; histamine 10 µg/kg; PGF₂ 50 µg/kg; PDBu, a PKC-activating phorbol ester, 5 µg/kg; PDD, an inactive phorbol ester, 5 µg/kg; and Bay K 8644, a dihydropyridine-sensitive L-type Ca²⁺ channel agonist, 3 and 10 µg/kg.^{13 14} In a preliminary study, we confirmed that the coronary hyperconstrictive responses to serotonin, histamine, PDBu, and Bay K 8644 were all reproducible in vivo. Those responses were examined before and after the intracoronary administration of staurosporine, a PKC inhibitor, 10 µg/kg (n=5, protocol 1); sphingosine, a PKC inhibitor, 100 µg/kg (n=5, protocol 2); ryanodine, an inhibitor of Ca²⁺-induced Ca²⁺ release, 1 µg/kg (n=5, protocol 3)^{10 15} ; thapsigargin, an inhibitor of Ca²⁺-ATPase of SR, 1 µg/kg (n=5, protocol 4)¹⁶ ; and nifedipine, a dihydropyridine-sensitive L-type Ca²⁺ channel antagonist, 100 µg/kg (n=5, protocol 5). In the protocol with PDBu, we started to administer the above-mentioned inhibitors after confirming that the coronary diameter returned to the control level.

Coronary Arteriography
The animals were anesthetized and ventilated as described above, and selective coronary arteriography was performed. A preshaped Judkins catheter was inserted into either the right or left femoral artery, and then coronary arteriography in a left anterior oblique view was performed under control conditions and after intracoronary nitroglycerin 10 µg/kg. ECGs (leads I, II, III, V₁, and V₆), along with the mean arterial pressure and heart rate, were recorded continuously during the experiments. Coronary arteriography was repeated 2 minutes after the intracoronary administration of serotonin and Bay K 8644; 1 minute after the administration of histamine; 5, 10, 20, 40, and 60 minutes after PDBu; 5, 10, and 20 minutes after PDD; and 5 minutes after PGF₂.^{8 9 11} We have previously confirmed that at these time points, coronary vasoconstricting responses peaked to the above vasoconstrictor agents.^{8 9 11} Ischemic ECG changes were defined as ST-segment elevation or depression >1 mm in more than two leads.^{8 11}

Coronary Diameter Measurement
Cinefilms were reviewed simultaneously by more than two observers, angiograms at end diastole were chosen and printed, and the coronary luminal diameters were measured with calipers.^{8 9 11} With this technique, excellent correlations between repeated measurements (r=.99) and between different observers (r=.98) were confirmed in the range of coronary diameters from 0.98 to 5.58 mm.^{8 9 11} The degree of constrictive response was expressed as the percent decrease in the luminal diameter from the level after intracoronary nitroglycerin 10 µg/kg. The coronary diameter was measured at the segments treated with IL-1ß beads and control beads and at the untreated segment at a comparable diameter.

Histological Examination
After the angiographic experiments, the hearts were removed, and the left coronary arteries were perfused via a constant-pressure perfusion system (120 mm Hg) with saline (500 mL) and subsequently with 6% formaldehyde (1000 mL).^{8 9} After fixation, both the left anterior descending and the left circumflex coronary arteries were cut transversely into segments at 5-mm intervals along their main trunks with small portions of surrounding tissue. These segments were then stained with hematoxylin-eosin and van Gieson's elastic stains for photomicroscopy. The intimal and medial areas of the coronary artery were measured in photomicrographs with a computer-assisted picture analysis system (Genlocker System, Sony Inc). The degree of the intimal thickening was expressed by the following three parameters: intimal area (in square millimeters), maximal intimal thickness (in millimeters), and percent intima. The latter was defined as the intimal area expressed as a percentage of the area delineated by the internal elastic lamina.^{8 9}

Drugs
The following drugs were used: 5-hydroxytryptamine (serotonin), histamine, PGF₂, PDD, PDBu, staurosporine, sphingosine, ryanodine, and thapsigargin (Sigma Chemical Co), Bay K 8644 (Wako Junyaku Co), and nifedipine (Bayer Pharmaceutical Co). PDD, PDBu, staurosporine, sphingosine, ryanodine, thapsigargin, and Bay K 8644 were prepared as stock solutions in DMSO 0.1%. The intracoronary administration of DMSO alone did not change the coronary diameter. Dilution was done with a physiological salt solution. Nifedipine was prepared by diluting the contents of a nifedipine capsule (10 mg) in 10 mL of physiological salt solution.

Statistical Analysis
The results were expressed as mean±SEM. Throughout the text, n represents the number of animals tested. A repeated-measures ANOVA was performed to evaluate global statistical significance, and if a significant F value was found, Scheffe's test was performed to identify the difference among the groups. Paired data of basal coronary diameter obtained from the IL-1ß-treated and the untreated segments were analyzed by Student's t test. A value of P<.05 was considered to be statistically significant.

	Results

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Hemodynamic Variables
The mean aortic pressure and heart rate did not change significantly during the experiments in any of the protocols (Table 1).

View this table: [in this window] [in a new window]   Table 1. Effects of PKC Inhibitors, Ryanodine, Thapsigargin, and Nifedipine on Heart Rate, Mean Arterial Pressure, and Coronary Artery Diameter

Induction of Coronary Artery Spasm
Control coronary angiograms showed that a mild stenotic lesion developed at the surgically treated site, which was greater at the IL-1ß-treated site (26±4%) than at the control bead–treated site (16±5%) (n=7, P<.05). Thus, the surgically treated site was easily identified by the presence of this stenotic lesion.

Intracoronary serotonin 10 µg/kg or histamine 10 µg/kg repeatedly induced coronary artery spasm at the site treated with IL-1ß beads, whereas only mild vasoconstriction was induced both at the site treated with control beads and at the untreated site (Fig 1). In contrast, PGF₂ 50 µg/kg induced comparable degrees of vasoconstriction at those three sites (Fig 1). These results confirmed our previous findings in the same model.^{8 9}

View larger version (135K): [in this window] [in a new window]   Figure 1. Coronary angiograms of the left coronary artery. Control (A) and after the intracoronary administration of serotonin 10 µg/kg (B), histamine 10 µg/kg (C), PDBu 5 µg/kg (D), Bay K 8644 10 µg/kg (E), and PGF2 50 µg/kg (F). Solid and open arrows indicate the IL-1ß–treated and the control bead–treated sites, respectively.

PDBu 5 µg/kg also induced coronary artery spasm at the IL-1ß–treated site (Fig 1). Fig 2A shows the time course of the PDBu-induced coronary vasoconstriction. At the IL-1ß–treated site, PDBu 5 µg/kg induced a marked coronary vasoconstriction that peaked at 10 to 20 minutes after intracoronary administration (56±4%). On the other hand, PDBu-induced coronary vasoconstrictions at the control bead–treated and the untreated sites were mild and the extents of the vasoconstrictions were comparable (34±4% and 25±2%, respectively). In contrast, PDD 5 µg/kg did not cause any significant coronary vasoconstriction at the IL-1ß–treated site (Fig 2B). Intracoronary administration of Bay K 8644 10 µg/kg caused coronary spasm at the IL-1ß–treated site (Fig 1). The Bay K 8644–induced coronary spasm at the IL-1ß–treated site was dose dependent, whereas the coronary vasoconstrictions induced by Bay K 8644 at the control bead–treated and the untreated sites were very mild and the extents of the vasoconstrictions were comparable (Fig 3). The incidence of ischemic ECG changes during the occurrence of coronary spasm was 28% to serotonin (7/25), 20% to histamine (5/25), 48% to PDBu (12/25), and 16% to Bay K 8644 (4/25) [overall incidence was 56% (14/25)].

View larger version (20K): [in this window] [in a new window]   Figure 2. A, Time course of the PDBu 5 µg/kg–induced coronary vasoconstriction at the IL-1ß–treated, control bead–treated, and untreated sites. PDBu induced marked coronary vasoconstriction at the IL-1ß–treated site, whereas only mild vasoconstriction was induced at the control bead–treated and the untreated sites. B, Time course of coronary vasoconstriction at the IL-1ß–treated site induced by PDBu 5 µg/kg, PDD 5 µg/kg, PDBu 5 µg/kg after pretreatment with staurosporine 10 µg/kg, sphingosine 100 µg/kg, nifedipine 10 µg/kg, ryanodine 1 µg/kg, and thapsigargin 1 µg/kg.

View larger version (17K): [in this window] [in a new window]   Figure 3. Bay K 8644–induced coronary vasoconstriction.

Effect of PKC Inhibitors on Coronary Vasomotion
The intracoronary administration of staurosporine 10 µg/kg and sphingosine 100 µg/kg caused slight and comparable degrees of coronary vasodilatation at the IL-1ß–treated site (4±2% and 3±2%, respectively) and the untreated site (4±3% and 5±3%, respectively) (Table 1). PDBu-induced coronary vasoconstrictions at the IL-1ß–treated site and the untreated site were significantly suppressed by pretreatment with staurosporine and sphingosine (Figs 2, 4, and 5). The coronary vasospastic responses to serotonin, histamine, and Bay K 8644 at the IL-1ß–treated site were also suppressed by the same doses of staurosporine and sphingosine, but the vasoconstriction at the untreated site was not affected by those PKC inhibitors (Figs 4 and 5). In contrast, pretreatment with intra-coronary administration of staurosporine or sphingosine did not affect the PGF₂-induced coronary vasoconstriction at either site (Figs 4 and 5).

View larger version (25K): [in this window] [in a new window]   Figure 4. Coronary vasoconstriction induced by serotonin 10 µg/kg (white hatched columns), histamine 10 µg/kg (black hatched columns), PDBu 5 µg/kg (stippled columns), Bay K 8644 10 µg/kg (solid columns), and PGF2 50 µg/kg (open columns) at the IL-1ß–treated (left) and the untreated (right) sites before and after pretreatment with staurosporine 10 µg/kg. *P<.05 vs before staurosporine; P<.05 vs IL-1ß–treated site.

View larger version (27K): [in this window] [in a new window]   Figure 5. Coronary vasoconstriction induced by serotonin 10 µg/kg (white hatched columns), histamine 10 µg/kg (black hatched columns), PDBu 5 µg/kg (stippled columns), Bay K 8644 10 µg/kg (solid columns), and PGF2 50 µg/kg (open columns) at the IL-1ß–treated (left) and untreated (right) sites before and after pretreatment with sphingosine 100 µg/kg. *P<.05 vs before sphingosine; P<.05 vs IL-1ß–treated site.

Effect of Ryanodine on Coronary Vasomotion
The intracoronary administration of ryanodine 1 µg/kg did not affect the basal coronary diameter at either the IL-1ß–treated or the untreated sites (Table 1). Pretreatment with ryanodine did not affect the coronary spasm induced by serotonin, histamine, PDBu, or Bay K 8644 at the IL-1ß–treated site (Fig 6). In contrast, the PGF₂-induced coronary vasoconstriction was significantly inhibited by pretreatment with ryanodine at both sites (Fig 6).

View larger version (31K): [in this window] [in a new window]   Figure 6. Coronary vasoconstriction induced by serotonin 10 µg/kg (white hatched columns), histamine 10 µg/kg (black hatched columns), PDBu 5 µg/kg (stippled columns), Bay K 8644 10 µg/kg (solid columns), and PGF2 50 µg/kg (open columns) at the IL-1ß–treated (left) and the untreated (right) sites before and after pretreatment with ryanodine 1 µg/kg. *P<.05 vs before ryanodine; P<.05 vs IL-1ß–treated site.

Effect of Thapsigargin on Coronary Vasomotion
The intracoronary administration of thapsigargin 1 µg/kg did not affect the basal coronary diameter at either the IL-1ß–treated or the untreated sites (Table 1). Pretreatment with thapsigargin did not affect the coronary spasm induced by serotonin, histamine, PDBu, or Bay K 8644 at the IL-1ß–treated site (Fig 7). In contrast, the PGF₂-induced coronary vasoconstriction was significantly inhibited by pretreatment with thapsigargin (Fig 7).

View larger version (29K): [in this window] [in a new window]   Figure 7. Coronary vasoconstriction induced by serotonin 10 µg/kg (white hatched columns), histamine 10 µg/kg (black hatched columns), PDBu 5 µg/kg (stippled columns), Bay K 8644 10 µg/kg (solid columns), and PGF2 50 µg/kg (open columns) at the IL-1ß–treated (left) and the untreated (right) sites before and after pretreatment with thapsigargin 1 µg/kg. *P<.05 vs before thapsigargin; P<.05 vs IL-1ß–treated site.

Effect of Nifedipine on Coronary Vasomotion
The intracoronary administration of nifedipine 100 µg/kg caused coronary vasodilatation at the IL-1ß–treated (4±2%) and the untreated (3±2%) sites (Table 1). The Bay K 8644–induced coronary vasoconstrictions at the IL-1ß–treated site and the untreated site were markedly inhibited by pretreatment with nifedipine (Fig 8). The coronary vasospastic responses to serotonin, histamine, and PDBu at the IL-1ß–treated site also were significantly suppressed by pretreatment with the same dose of nifedipine, but the vasoconstriction at the untreated site did not change after pretreatment with nifedipine (Fig 8). The PGF₂-induced coronary vasoconstriction at both sites was also suppressed by pretreatment with nifedipine (Fig 8).

View larger version (27K): [in this window] [in a new window]   Figure 8. Coronary vasoconstriction induced by serotonin 10 µg/kg (white hatched columns), histamine 10 µg/kg (black hatched columns), PDBu 5 µg/kg (stippled columns), Bay K 8644 10 µg/kg (solid columns), and PGF2 50 µg/kg (open columns) at the IL-1ß–treated (left) and the untreated (right) sites before and after pretreatment with nifedipine 100 µg/kg. *P<.05 vs before nifedipine; P<.05 vs IL-1ß–treated site.

Table 2 summarizes the present results.

View this table: [in this window] [in a new window]   Table 2. Summary of Vasoconstrictor Responses in the Present Study

Histology
At the IL-1ß–treated site, significant intimal thickening (intimal area, 0.13±0.03 mm²; maximal intimal thickness, 0.17±0.04 mm; % intima, 15±7%; n=7) was noted. At the control bead–treated site, in contrast, the intimal thickening was minimal (intimal area, 0.05±0.01 mm²; maximal intimal thickness, 0.04±0.01 mm; % intima, 6±5%; n=7), and no intimal thickening was noted at the untreated site, as reported previously.^{8 9}

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrated the following novel findings on the intracellular mechanisms responsible for coronary spasm in our new swine model in vivo (Table 2): (1) PDBu, a PKC-activating phorbol ester, induced coronary spasm at the IL-1ß–treated inflammatory/proliferative lesion, where serotonin and histamine also induced spasm; (2) Bay K 8644, a dihydropyridine-sensitive L-type Ca²⁺ channel agonist, also induced coronary spasm at the IL-1ß–treated site; (3) two kinds of PKC inhibitors, staurosporine and sphingosine, inhibited the coronary spasm induced by serotonin, histamine, PDBu, and Bay K 8644 but had no effect on the PGF₂-induced vasoconstriction; (4) ryanodine, an inhibitor of Ca²⁺-induced Ca²⁺ release, and thapsigargin, an inhibitor of Ca²⁺-ATPase of SR, did not inhibit the coronary vasospasm induced by serotonin, histamine, PDBu, or Bay K 8644 but did inhibit the coronary vasoconstriction induced by PGF₂; and (5) nifedipine, a dihydropyridine-sensitive L-type Ca²⁺ channel antagonist, suppressed the vasoconstriction to all agonists tested.

Coronary Spasm at the Inflammatory/Proliferative Lesion
On the basis of the pathological finding that adventitial inflammatory lesions were noted at the spastic site of the coronary artery in patients with active variant angina,^{17 18} we attempted to determine what morphological and functional changes of the coronary artery were induced by chronic adventitial treatment with IL-1ß, a major inflammatory cytokine found in the atherosclerotic lesions.^{19 20} The treatment resulted in proliferative changes and vasospastic responses of the coronary artery.^{8 9} The inflammatory/proliferative coronary segments exhibited a typical hyperreactivity to some vasoconstrictor agents (serotonin, histamine, PDBu, and Bay K 8644), since they constricted more than control segments at the same dose, hence meeting the definition proposed by Maseri et al.²¹ We previously confirmed that in our new swine model, the spastic activity lasts for at least 3 months.⁸ Since the spasm in our new swine model exhibited many similarities to that in our original model,^{5 6 7} whose activity lasts for more than a year,¹¹ we consider that the spastic activity in our new model may also last for a longer period. Thus, our new swine model of coronary spasm exhibits some similarities to coronary spasm in humans and suggests that the inflammatory/proliferative changes of the coronary artery may play an important role, at least in part, in the occurrence of the spasm in humans. Using this model, we examined the intracellular mechanisms of coronary spasm in vivo, while paying special attention to the two important intracellular mechanisms, Ca²⁺ release from the SR and PKC-mediated pathway.^{10 11}

PDBu-Induced Coronary Artery Spasm
The present study demonstrated that PDBu, a PKC-activating phorbol ester, induced vasoconstriction in the porcine coronary arteries in vivo, and the vasoconstriction was significantly augmented at the IL-1ß–treated site over the control site. This is consistent with our previous finding in the original model of coronary spasm.¹¹ The PDBu-induced coronary spasm was blocked by pretreatment with two kinds of PKC inhibitors with different mechanisms of action: staurosporine binds to the catalytic site of PKC, and sphingosine binds to the regulatory site.²² An inactive phorbol ester, PDD, did not induce any coronary vasoconstriction. These results indicate that PKC-mediated responses were augmented at the inflammatory/proliferative lesion in our swine model in vivo.

However, the mechanism for phorbol ester–induced vasocontraction is still unknown. Fish et al²³ reported that 12-O-tetradecanoyl phorbol-13-acetate increases the dihydropyridine-sensitive Ca²⁺ conductance in the A7r5 rat aortic smooth muscle cell line. Hirakawa et al²⁴ also recently reported that PDBu increased the dihydropyridine-sensitive Ca²⁺ conductance in rat aortic smooth muscle cells in primary culture. Other investigators have also reported that phorbol ester–induced vasoconstriction is not accompanied by a change in the intracellular Ca²⁺ level,^{25 26} which implies that Ca²⁺ sensitivity of the contractile proteins may be augmented by activation of PKC with phorbol esters.

If PKC is tonically activated at the IL-1ß–induced inflammatory/proliferative lesion under basal conditions, a smaller response to PDBu (because of the reduced availability of PKC) and a greater vasodilation to staurosporine would be expected at the IL-1ß–treated site. However, this was not the case in our study. PKC-mediated sustained vasoconstriction may play a role in cerebral vasospasm after subarachnoid hemorrhage, when PKC is tonically activated under basal conditions and the spontaneous, diffuse vasospasm lasts for days to weeks.²⁷ We consider that the mass of and/or the substrate for PKC may be increased at the inflammatory/proliferative lesion and that PKC-mediated responses are augmented only on stimulation but not under basal conditions.¹¹

PKC-Mediated Pathway in Serotonin- and Histamine-Induced Coronary Artery Spasms
In the present study, coronary hyperconstrictions induced by serotonin and histamine at the IL-1ß–treated site were significantly inhibited by pretreatment with PKC inhibitors. This indicates that the PKC-mediated pathway contributes substantially to the pathogenesis of coronary spasm to the autacoids in our new swine model. These results are consistent with our previous findings, in which it was demonstrated that the PKC-mediated pathway plays an important role in coronary spasm in our original model,¹¹ as well as in the hyperconstriction of vascular smooth muscle from Watanabe heritable hyperlipidemic rabbits.²⁸ Interestingly, however, the PGF₂-induced coronary vasoconstriction at the IL-1ß–treated and the control sites was not affected by pretreatment with the PKC inhibitors. This suggests that the intracellular signaling that contributes to the PGF₂-mediated vasoconstriction is regulated primarily by mechanism(s) other than the PKC-mediated mechanism in vivo. The results with PGF₂ also suggest that the concentrations of staurosporine and sphingosine used in the present study were relatively selective for PKC inhibition.

Bay K 8644–Induced Coronary Artery Spasm
Bay K 8644, an agonist of dihydropyridine-sensitive L-type Ca²⁺ channel, also induced coronary spasm at the IL-1ß–treated site in the present study. The finding that Bay K 8644–induced spasm was significantly inhibited by pretreatment with PKC inhibitors suggests the important role of the PKC-mediated pathway in the Ca²⁺ channel agonist–induced spasm. The activation of PKC increases the dihydropyridine-sensitive Ca²⁺ conductance in a vascular smooth muscle cell line.^{23 24} In our original swine model, in which we also demonstrated the occurrence of Bay K 8644–induced coronary spasm,¹¹ no difference was observed in the calcium-tension relation of the contractile proteins in the skinned arterial strips between the spastic and the control sites, which thus indicated that Ca²⁺ sensitivity of the contractile proteins per se is not augmented at the spastic site.²⁹ Thus, the present results suggest that the Ca²⁺ influx through dihydropyridine-sensitive L-type Ca²⁺ channel and/or Ca²⁺ sensitivity of the contractile proteins may be augmented via the PKC-mediated pathway.

Role of Intracellular Ca²⁺ Pool
Another important finding of the present study was that PGF₂ induced comparable degrees of coronary vasoconstriction at the spastic and the control sites. This finding was also observed repeatedly in our previous studies.^{8 9} Phenylephrine⁶ and a thromboxane A₂ analogue³⁰ also caused comparable degrees of coronary vasoconstriction at the spastic and the control sites in our original swine model. These results may not only rule out the major contribution of the geometric theory³¹ but also suggest that coronary hyperreactivity develops selectively to some agonists but not to others, probably because of the altered intracellular signal transduction mechanism(s). Recently, it was also reported that in the rat basilar artery, PKC mediates vasoconstrictions to serotonin but not those to PGF₂ in vivo.³²

In the present study, ryanodine (an inhibitor of the Ca²⁺-induced Ca²⁺ release from the SR store^{10 15} ) and thapsigargin (an inhibitor of Ca²⁺-ATPase of the SR¹⁶ ), both of which deplete the SR Ca²⁺ store, significantly inhibited the vasoconstriction of the porcine coronary artery caused by PGF₂ but did not inhibit the coronary spasm induced by serotonin, histamine, PDBu, or Bay K 8644. These results indicate that the local coronary hyperreactivity is not generalized to all constrictor stimuli and suggest that the PKC-mediated pathway is important for the vasospastic responses induced by serotonin, histamine, PDBu, and Bay K 8644, whereas the Ca²⁺ release from the SR into the cytosol may be important for the vasoconstriction induced by PGF₂ in vivo (Table 2). We also recently observed that in the aortas of hypercholesterolemic rabbits, the hyperreactivity to serotonin was mediated by PKC but the constriction induced by PGF₂ was not inhibited by PKC inhibitor.²⁸ Furthermore, Murray et al³² reported that the vasoconstriction evoked by serotonin but not that by PGF₂ was inhibited by PKC inhibitors in the rat basilar artery in vivo. In addition to those previous findings, we demonstrate in the present study that the coronary vasoconstricting responses were augmented only in response to the agonists whose effect depends on the PKC-mediated pathway but not to those whose effect depends primarily on the intracellular Ca²⁺ pool. Further studies are needed to clarify the intracellular mechanism(s) for the selective hyperreactivity at the spastic site.

In summary, the present study demonstrated that the PKC-mediated pathway is substantially involved in the pathogenesis of coronary artery spasm at inflammatory/proliferative lesions induced by chronic treatment with IL-1ß. In contrast, the Ca²⁺ release from the SR into the cytosol may not play a primary role in the pathogenesis of coronary spasm in our new swine model. It remains to be examined to what extent the enhanced PKC-mediated pathway is involved in the long-lasting local hyperreactivity in patients with variant angina.

	Acknowledgments

 
This work was supported in part by grants 02404045, 04670540, 05454274, and 07457173 from the Ministry of Education, Science, and Culture, Tokyo, Japan; grant 6C-2 from the Ministry of Health and Welfare, Tokyo, Japan; a grant-in-aid from the Sandoz Foundation Gerontological Research, Basel, Switzerland; the Japan Research Foundation for Clinical Pharmacology, Tokyo, Japan; and the Japanese Medical Association, Tokyo, Japan. The authors wish to thank Drs Masato Hirata and Junji Nishimura, Kyushu University, for valuable comments on the manuscript and Mika Mizokami and Tomoko Takebe for their technical assistance.

	Selected Abbreviations and Acronyms

 

IL-1ß = interleukin-1ß IP3 = inositol 1,4,5-triphosphate PDBu = phorbol-12,13-dibutyrate PDD = phorbol-12,13-didecanoate PGF2 = prostaglandin F2 PKC = protein kinase C SR = sarcoplasmic reticulum

	Footnotes

 
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.

Received November 6, 1995; revision received January 4, 1996; accepted January 7, 1996.

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