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Increased constrictor response to acetylcholine of the isolated coronary arteries from patients with variant angina
ELSEVIER
International
Journal of Cardiology
52 (1995) 223-233
Increased constrictor response to acetylcholine of the isolated coronary arteries from patients with variant angina Kiyotaka Kugiyama *, Toyoaki Murohara, Hirofumi Yasue, Tadashi Kimura, Naritsugu Sakaino, Masamichi Ohgushi, Seigo Sugiyama, Ken Okumura Division of Cardiology, Kumamoto University School of Medicine, Honjo I-1-1, Kumamoto City 860, Japan Received 27 June 1995; revision received 1 August 1995; accepted 31 August 1995
Abstract The aim of this study was to determine whether isolated coronary arteries from patients with variant angina show hyperreactivity and/or supersensitivity to acetylcholine in vitro. Coronary arterial rings were obtained at autopsy within 3 h after death from six coronary arteries having spasmin four patients with variant angina and from 22 coronary arteries in 14 control patients with non-cardiac death. The coronary rings were suspendedin the organ chamber filled with Krebs Henseleit solution bubbled with 95% 0, + 5% CO,, and their isometric tensions were monitered. Arterial rings isolated from both the patients with variant angina and the controls contracted dose-dependently in response to acetylcholine (10-9-10-5 mol/l). EC,, of acetylcholine (i.e. concentration producing 50% of maximum contraction) was not significantly different between the coronary arteries from patients with variant angina and those from controls, but maximum contraction elicited by acetylcholine (expressedas a percentageof the contraction elicited by 60 mmoyl KCl) was significantly greater in the coronary arteries from patients with variant angina than those from controls. In conclusion, the isolated coronary arteries from patients with variant angina have hypercontractile reactivity to acetylcholine. This intrinsic alteration of the coronary reactivity to acetylcholine may play a role in the genesis of coronary spasm occurring in the situations of enhanced parasympathetic nervous discharge. Keywords: Coronary artery spasm;Variant angina; Acetylcholine
1. Introduction It has been shown that coronary artery spasm plays an important role in the wide spectrum of ischemic heart disease [l-3]. Coronary spasm usually occurs at rest, particulary from midnight * Corresponding author, Tel.: +81 96 3442111 (Ext 5872); Fax: +81 96 3623256.
to early morning when the activity of the parasyrnpathetic nervous system is enhanced [3,4]. We have demonstrated that subcutaneous injection of methacholine or intracoronary injection of acetylcholine, a neurotransmitter of the parasympathetic nervous system, induces coronary artery spasm in patients with variant angina, suggesting that parasympathetic nervous system may play an important role in the mechanism(s) and the circadian
0167-5273/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-5273(95)02478-F
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variation of coronary artery spasm [3-81. However, the precise mechanism whereby acetylcholine induces coronary artery spasm in patients with variant angina still remains unknown. Acetylcholine is known to have two distinct effects on vascular tension; one is direct vascular smooth muscle contraction, and the other is endothelium-dependent vasorelaxation [9- 111.Therefore, at least two possible mechanisms of acetylcholine-induced coronary spasmare suggested:(1) a hyperreactivity of the coronary vascular smooth muscle to acetylcholine; (2) an impaired endothelium-dependent vasodilator response to acetylcholine. Furthermore, it also remains to be determined which factors may importantly contribute to the genesis of coronary artery spasm, neurohumoral abnormalities or intrinsic functional alterations of coronary arteries. The in-vivo studies seem to have difficulty in providing conclusive answersfor these questions. Therefore, a study examining vasomotor regulation of the isolated coronary arteries from patients with variant angina is required to determine the mechanism(s) of coronary spasm. Autopsy casesof patients with variant angina are rare, and to our knowledge, there has been no report examining the responseto acetylcholine of the isolated coronary arteries from patients with variant angina. In this study, we examined the effects of acetylcholine on the tonus of isolated coronary arteries from the patients with variant angina in comparison with those from the controls.
cf Cardiology 52 (1995) 223-233 2. Materials and methods
Coronary arterial segmentsat the spastic sites were isolated at autopsy within 3 h after death from six coronary arteries having spasm in four patients with variant angina. The coronary segmentswere also isolated at autopsy within 3 h after death from the mid portion of 22 coronary arteries in 14 patients who had no apparent cardiac diseaseduring their life, serving as controls. 2.1. Patients
A summary of clinical characteristics and cause of death of the patients with variant angina is shown in Table 1. All of the patients had attacks of chest pain associatedwith ST segmentelevation on the electrocardiograms (occurred at rest, usually in the middle of the night or early morning). Coronary artery spasm was angiographically demonstrated in all of the patients during their life at the spontaneous attack or the attack induced by intracoronary injection of acetylcholine [5-81. The angina1 attacks in all of the patients were almost completely suppressedby anti-angina1 drugs which had been discontinued for more than 3 days before death becauseof inability to take orally. All patients with variant angina had no myocardial infaction. Informed consent for cardiac catheterization was obtained from each patient. The causes of death in 14 control patients (52-68 years old, mean age 59, men) were hepato-
Table 1 Clinical characteristics of patients with variant angina Case
Age/Sex Cause of death
ST elevation during attacks
Sites of spasm
Induction of spasm Organic stenosis LAD LCX RCA
1 (F.T.)
64/M
Cholangiocarcinoma
11,111,aVF
2 (K.Y.) 57/M 3 (T.M.) 65/M
Hepatoeellular carcinoma Vt-s Hepatocellular carcinoma II, III, aVF, VI-s
4 (T.V.)
Grawitz’s tumor
52/M
II, III, aVF
RCA, LAD, LAD, RCA, LAD, RCA,
mid proximal proximal proximal proximal mid
ACh (50 pd ACh (50 ml ACh (100 ag) Spontaneous Spontaneous ACh (50 rg)
N
N
25%
N N
N N
N N
75% 75% 90%
Coronary artery spasmwas angiographically demonstrated at the spontaneousattack or the attack induced by intracoronary injection of acetylcholine during their life. Organic stenosis was evaluated after sublingual administration of nitroglycerin. Age, age at death; ST, ST segmentin electrocardiographic leads; LAD, left anterior descendingcoronary artery; LCX, left circumflex coronary artery; RCA, right coronary artery; Mid or proximal, mid or proximal portion of the coronary artery; M, male; N, no organic stenosis.
K. Kugiyama et al. /International
cellular carcinoma (four patients), adenocarcinoma of the stomach (four patients), adenocarcinoma of the colon (four patients), transitional cell cancer of the bladder (one patient), and glioblastoma multiforme (one patient). 2.2. Coronary arteriography in patients with variant angina (Table I) Case 1 (F. T.). The left coronary artery was nor-
mal, and the right coronary artery had a slight nar-
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rowing in the middle position at baseline. Injection of acetylcholine chloride (50 pg; approximately 5 pmol/l equivalent in the left coronary blood circulation) into the left coronary artery induced a subtotal occlusion at the proximal site of the left anterior descending coronary artery, which was associatedwith chest oppression and ST segment depression on the electrocardiographic leads V,-,. The spasmof the left anterior descendingcoronary artery resolved spontaneously. Thereafter, acetyl-
B
.
CASE
1 (RCA1
0
CONTROL
ACETYLCHOLINE.
-log M
Fig. 1. Case 1 (the right coronary artery) in Table 1. Coronary arteriograms showing acetylcholine-induced spasm at cardiac catheterization during their life and in-vitro responses to acetylcholine of the isolated right coronary artery at the site of spasm. (A) The right coronary artery at baseline. (B) Intracoronary injection of acetylcholine (50 pg) induced diffuse spasm (indicated by arrows), which was associated with chest oppression and ST segment elevation on electrocardiographic leads II, III, and aVF. (C) Spasm of the right coronary artery was resolved by intracoronary injection of isosorbide dinitrate. (D) Dose-response curves for acetylcholineinduced contraction of the isolated coronary arteries in vitro. Values in Case 1 are the mean f S.E.M. of the responses to acetylcholine of the four rings prepared from the right coronary artery at the site of the spasm. Values in Control are the mean f S.E.M. of the responses of the 22 rings from the isolated coronary arteries from 14 control patients who had no apparent cardiac disease. Contractions are expressed as a percentage of the contraction elicited by 60 mmovl KCL. -log M = -log [moliI].
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choline chloride (50 pg; approximately 10 pmol/l equivalent in the right coronary blood circulation) was again injected into the right coronary artery and induced a diffuse spasmat the mid position of the right coronary artery, which was associated with chest oppression and ST segmentelevation on electrocardiographic leads II, III, and aVF. The spasmof the right coronary artery was resolved by intracoronary injection of isosorbide dinitrate (Fig. 1). Case2 (K. Y.). The left coronary artery was normal at baseline. Intracoronary injection of acetyl-
Journal of Cardiology 52 (1995) 223-233
choline chloride (100 pg; approximately 10 pmol/l equivalent in the left coronary blood circulation) induced total occlusion at the proximal site of the left anterior descending artery (indicated by an arrow), which was associated with chest oppression and ST segment elevation on electrocardiographic leads Vi+ Spasm of the left anterior descendingcoronary artery was resolved by injection of isosorbide dinitrate into the left coronary artery (Fig. 2). Case3 (TM). Both of the left and right coronary arteries were normal at baseline. Simulta-
Fig. 2. Case 2 in Table 1. (A) Left coronary artery at baseline. (B) Intracoronary injection of acetylcholine (100 pg) induced total occlusion (indicated by an arrow), which was associatedwith chest oppression and ST segmentelevation on electrocardiographic leads VI-,. (C) Spasm of the left coronary artery was resolved by injection of isosorbide dinitrate into the left coronary artery. (D) Doseresponse curves for acetylcholine-induced contraction of the isolated coronary arteries in vitro. Values in Case 2 are the mean * S.E.M. of the responsesto acetylcholine of the four rings prepared from the left anterior descendingcoronary artery at the site of the spasm. Values in Control are same as shown in Fig. 1.
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Journal of Cardiology 52 (1995) 223-233
neous spasm of the right coronary artery (total occlusion) and the left anterior descending coronary artery (subtotal occlusion) occurred spontaneously and was associated with chest oppression and ST segment elevation on the electrocardiographic leads II, III, aVF, and Vim4. A fall in blood pressure and ventricular tachycardia occurred during the spasm.The spasmwas resolved by injection of isosorbide dinitrate into both of the coronary arteries (Figs. 3,4).
227
Case 4 (T. U.). The organic stenoseswere found in the left descending coronary artery (75% stenosis),the left circumflex coronary artery (75% stenosis), and the right coronary artery (90% stenosis). Injection of acetylcholine chloride (50 pg) into the right coronary artery induced a subtotal occlusion just distal to the organic stenosisof the right coronary artery, which was associated with typical substernal chest pain and ST segment elevation on the electrocardiographic leads II, III,
A
6 jz
loo-
.
CASE 3 IRCAl
0
CONTROL
z 5 0
50-
S
Fig. 3. Case3 (the right coronary artery) in Table I. (A) The right coronary artery at baseline. (B) Total occlusion of the right coronary artery (indicated by an arrow) occurred spontaneously, and spasm of the left anterior descending coronary artery also occurred simultaneously. Chest oppression and ST segmentelevation on electrocardiographic leads II, III, aVF, and Vl-, were associated.(C) Spasmof the right coronary artery was resolved by the intracoronary injection of isosorbide dinitrate. (D) Dose-responsecurves for acetylcholine-induced contraction of the isolated right coronary arteries in vitro. Values in Case 3 are the mean f S.E.M. of the responsesto acetylcholine of the four rings from the right coronary artery at the site of the spasm. Values in Control are same as shown in Fig. 1.
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al. /International
Journal of Cardiology 52 (1995) 223-233
ACETYLCHOLINE.
-log M
Fig. 4. Case 3 (the left anterior descending coronary artery) in Table 1. Coronary arteriograms during coronary spasm in this case was also described in the legend of Fig. 3. (A) The left coronary artery at baseline. (B) Spasmof the left anterior descendingcoronary artery occurred spontaneously. (C) Spasm was resolved by the intracoronary injection of isosorbide dinitrate. (D) Dose-response curves for acetylcholine-induced contraction of the isolated left anterior descendingcoronary arteries in vitro. Values m Case 3 are the mean f S.E.M. of the responsesto acetylcholine of the four rings from the left anterior descendingcoronary artery at the site of the spasm. Values in Control are same as shown in Fig. 1.
and aVF. The spasmwas resolved by injection of nitroglycerin into the right coronary artery. Coronary angiograms and histological findings of the coronary arteries in Case 4 in Table 1 have been previously reported [ 121. 2.3. Experimental procedure
Coronary segments(20 mm long) at the sites of the spasm in the isolated coronary arteries from patients with variant angina and those at the mid portion of the coronary arteries from the control patients were used for the muscle chamber experi-
ments and the histological examinations. A transverse section of the coronary segmentswas made, and the coronary segmentswere divided into two parts; one part was used for histological examinations and the other part was cleaned from connective tissues and cut into ring segments2 mm long. Each ring was vertically suspended between stainless steel hooks in an organ chamber with 10 ml of Krebs Henseleit solution (in mmol/l; NaCl 118, KC1 4.7, NaH2P04 1.2, MgSO4 1.2, CaCl* 2.0, NaHC03 25, EDTA 0.05, and glucose 10.0). The solution was bubbled with mixed gas (95%
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Journal of Cardiology 52 (1995) 223-233
O2 + 5% CO*) and maintained at 37°C and pH 7.4. The lower hook was anchored to the bottom of organ chamber, while upper one was connected to a force transducer UL30GR (Minebia, Tokyo, Japan), and isometric tension changes were recorded on an ink-writing pen recorder SR6211 (Graphtec, Tokyo, Japan). Rings were progressively stretched over 90 min to the optimum resting tension previously determined to be 4 g. The Krebs Henseleit solution was replaced every 20 min. After the equilibration period, the contractile responseto 60 mmol/l KC1 was examined first, then the rings were washed repeatedly and equilibrated for at least 30 min before the next protocol. The arterial rings that failed to produce > 1 g contraction in response to 60 mmoyl KC1 were considered to be invalid for the vascular smooth musclefunction and therefore were discarded from the study. Thereafter, increasing concentrations of acetylcholine were added cumulatively into the organ chamber. In one or two rings of each artery obtained from the patients with variant angina and the control patients, the increasing concentrations of acetylcholine were added cumulatively after precontraction with prostaglandin Fz, (30 pmol/l) in order to examine endothelium-dependent relaxation to acetylcholine. More than three rings were prepared from one coronary artery and the responseswere averaged and included in the final data analysis. 2.4. Histological examination
The segmentsof the coronary arteries were fixed in 10% formalin solution and embedded in paraffin, then sliced into sections of 3 pm thickness. Histological sections were stained with haematoxylin-eosin and Verhoeff-Van Gieson’s elastic stains. Prostaglandin FZa was obtained from Ono Pharmaceuticals (Osaka, Japan). Other chemicals were from Sigma Chemical (St Louis, MO). Data were expressedas mean f S.E.M. Statistical evaluation of the difference of two meanswas performed by unpaired Student’s t-test. When more than two means were compared, analysis of variance (ANOVA) followed by the Bonferroni’s t-test was used. Differences between values were
considered to be statistically significant when P < 0.05.
The study was in agreementwith the guidelines approved by the ethics committee at our institution. 3. Results
Acetylcholine constricted all coronary arterial rings from controls as well as from patients with variant angina (Fig. l-5). Constrictor responsesto acetylcholine (expressed as a percentage of contraction elicited by 60 mmol/l KCl) are summarized in Fig. 6. Maximal constrictor responses to acetylcholine were significantly greater in the coronary rings from the patients with variant angina than in those from the control patients (138 f 6.2% vs. 92 f 7.2%, n = 6 and 22, respectively, P < 0.01). The ECsOvalue of acetylcholine, the concentration producing 50% of maximum contraction, was not significantly different between the patients with variant angina and the controls (-log [mol/l]; 6.6 f 0.4 and 6.7 f 0.3, n = 6 and 22, respectively). Acetylcholine did not relax any arterial rings tested from both groups after precontraction with prostaglandin Fza. Relaxation induced by sodium nitroprusside (1 pmol/l) was completely preserved in all arterial rings from both groups.
CONTROL
VARIANT ANGlN,i
k;.--
:; ‘: $
\
I Fig. 5. Representative recordingsshowingthe contractile responses to acetylcholine in the isolatedcoronaryarteries from a patientwith variant angina and a control patient. Acetylcholine (ACh) was added to the muscle chamber to give the concentrations shown (expressed as -log [moM]). KCL, the contraction evoked by 60 mmoM KCI; SNP, sodium nitroprnsside (1 PmoyI); W, washing,
230
K. Kugiyama et al. /International 0 VARIANT ANGINA (n=6)
0 CONTROL (n=22) * p
Fig. 6. Dose-responsecurves for acetylcholine-induced contraction of the six isolated coronary arteries having spasm from four patients with variant angina and of the 22 isolated coronary arteries from 14 control patients. Contractions are expressedas a percentageof the contraction elicited by 60 mmohl KCI. -log M = -log [mobs].
3.1. Histological findings
Endothelial cell lining was completely preserved in all arterial sections from the patients with variant angina and the control patients. Intimal hyperplasia was observedin all arterial sections from the patients with variant angina as well as the controls. Intimal calcification and cholesterol crystal deposits were shown in the sections from the left anterior descendingcoronary artery in Case2 and from the right coronary artery in Case 4. No significantly abnormal changewas microscopically observed in media in all arterial sections from the patients with variant angina. There was no apparent abnormal finding in adventitia in the sections from Cases1,2, and 3, but scatteredinfiltration of inflammatory cells and degenerative and fibrotic perivascular autonomic nerves were found in the adventitia of the right coronary artery from Case 4 as reported previously [ 121. 4. Discussion
Coronary artery spasmoccurs most often when patients are at rest, particularly from midnight to early morning, and is usually not provoked by ex-
Journal of Cardiology 52 (1995) 223-233
ercisein the daytime [ 13,141.It is also well known that the activity of the parasympathetic nervous system is enhanced at rest and is suppressed by physical activity [ 15,161. Furthermore, we have shown that subcutaneous injection of methacholine or intracoronary injection of acetylcholine, the neurotransmitter of the parasympathetic nervous system, induces coronary artery spasm and that the attack can be suppressed by atropine, a parasympatholytic agent, in some patients with variant angina [4-71. We therefore postulated that the activity of the parasympathetic nervous system might be related to the pathogenesisand circadian variation of variant angina or coronary spasm. The present study demonstrated that the isolated coronary arteries from the patients with variant angina had greater constriction than those from the controls in response to acetylcholine at the concentrations of l-10 pmol/l, and these concentrations were the same as those infused into the coronary artery in vivo human for the induction of coronary artery spasm at cardiac catheterization [5-71. The present result indicates that the increased contractile responseof the coronary arteries to acetylcholine in patients with variant angina seems not to be caused by the neurohumoral factors but is intrinsic to the coronary arteries in these patients. This hyperreactivity to acetylcholine in the coronary arteries could play a role in the genesisof coronary spasm occurring in the condition of enhanced parasympathetic nervous discharge. This is in agreement with the phenomenon observed in the swine model of coronary spasm 1171.
Coronary artery spasm is angiographically defined as a total occlusion or a subtotal occlusion of the epicardial arteries leading to myocardial ischemia [l-7] and should be distinguished from the non-occlusive vasoconstriction observed in the atherosclerotic coronary arteries in humans [ 18-201 or experimentally endothelium-denuded arteries in animal models [lo]. The atherosclerotic coronary arteries might exhibit supersensitivity (samedegreeof diameter reduction as that in normal arteries occurs in responseto a lower dose of constrictor agonists), but the maximal reduction of arterial diameter is usually ~30% and is not enough to cause myocardial ischemia [6,18-211.
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Journal of Cardiology 52 (1995) 223-233
On the other hand, coronary arteries involved in spasm do not necessarily have hypersensitivity in response to the intracoronary infusion of the agonists but exhibit increased maximal contractile response (hyperreactivity) sufficiently to occlude arteries in angiographic studies [22-241. From this point of view, the present study shows that the isolated coronary arteries from patients with variant angina exhibit hyperreactivity (expressedas a maximal contraction) but not hypersensitivity (expressed as EC,,) in response to acetylcholine, which may be in agreementwith the characteristic contractile responsesof coronary arteries having spasm to intracoronary infusion of acetylcholine, observed in angiographic studies. The balance between two opposing muscarinic effects, endothelium-mediated vasorelaxation and direct stimulation of smooth muscle, determines vasomoter responses to acetylcholine [9- 1I]. Acetylcholine has been shown to cause endothelium-dependent relaxation in normal vessels of most spieces [9,11,25]. However, relaxation to acetylcholine was not shown in any coronary arteries tested from the patients with variant angina as well as the control patients in the present study, although histological examination showed complete preservation of endothelial lining of the coronary arteries from both groups. There are conflicting results in the muscarinic endotheliumdependent relaxation of the isolated human coronary arteries. Ginsburg [26] and other workers [27] reported that isolated human coronary arteries obtained from hearts of transplant recipients or cadavers lack the phenomenon of endothelium-dependent relaxation and constrict in response to acetylcholine. Bossaller et al. [25] however, showed that non-atherosclerotic coronary arteries from the hearts of transplant recipients exhibit muscarinic endothelium-dependent relaxation. It is now well-known that atherosclerosis impairs muscarinic endothelium-dependent relaxation [19-20,251 and begins in childhood and progresseswith increasing age [28,29]. In the present study, intimal thickening was present in the coronary arteries isolated from the patients with variant angina as well as those from the controls, which may cause defective muscarinic endothelium-dependent relaxation.
231
The observed hypercontractile responses to acetylcholine in coronary arteries from the patients with variant angina seem to have resulted from the enhanced responseof the arterial smooth muscle to acetylcholine. The precise mechanism for the hyperreactive contractility, without change of EC!sOvalues in response to acetylcholine, remains entirely unknown. The change of the receptor population and/or the post-receptor signalling pathway linked to the contractile protein in smooth muscle might be involved in the mechanism explaining for the present findings. Clinical studies [ 18-201 and in-vitro experiments [21,25,30] show that hypercholesterolemia and atherosclerosis enhance constrictor responses of arterial smooth muscle to various agonists. The present pathological examination showed substantial intimal thickening in coronary rings from patients with variant angina. Therefore, atherosclerosis may partly play a role in the mechanisms of the hypercontractile responseto acetylcholine in coronary arteries from patients with variant angina. However, hyperlipidemia is not a risk factor in patients with variant angina [31,32] and we have found that spasmoccurs often in angiographically normal coronary arteries, especially in patients with multivessel coronary artery spasm [7,33]. Therefore, other unknown mechanism(s)as well as atherosclerosis could contribute to the hypercontractility of the coronary arterial smooth muscle in response to acetylcholine in patients with variant angina. Coronary artery spasmcan be induced in patients with variant angina or miniture swine by various agonists such as ergonovine, dopamine, and histamine as well as acetylcholine [5,17,22,23,35-371.It remains undetermined in the present study whether the hyperreactive contractile responseof the isolated coronary arteries from patients with variant angina may be also shown to these agonists known to provoke coronary spasm. Yokoyama et al. [38] reported a case of variant angina whose post-mortem isolated coronary artery showed supersensitivity to ergonovine. Although they did not address the hyperreactivity against hypersensitivity, their data showed hyperreactivity to ergonovine of the isolated coronary artery from patients with variant angina. The enhanced responsivenessof the coronary artery to
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agonists with disparate surface receptors in patients with variant angina suggestsfunctional alteration(s) of the post-receptor regulatory pathway, shared by the different receptor stimulations, for smooth muscle contraction [24]. In conclusion, the isolated coronary arteries from patients with variant angina have the increased reactivity to acetylcholine. The hyperreactivity of the coronary arteries in response to acetylcholine could contribute to the coronary artery spasm occurring at rest when the activity of the parasympathetic nervous system is enhanced, supporting the hypothesis that parasympathetic nervous system may play an important role in the pathogenesis and the circadian variation of the attack of variant angina or coronary artery spasm. Acknowledgement This study was supported in part by grants-inaid for Scientific Research on Priority Area (03268107), B03454257, and C3670460 from the Ministry of Education, Science, and Culture in Japan and the Smoking Research Foundation, Tokyo, Japan. References [1] Hills LD, Braunwald E. Coronary artery spasm. N Engl J Med 1978;299: 695-702. [2] Maseri A, Severi S, DeNes M et al. Variant angina: one aspect of a continuous spectrum of vasospastic myocardial ischemia. Am J Cariol 1978;42: 1019-1035. [3] Yasue H, Omote S, Takizawa A, Nagao M. Coronary arterial spasmin ischemic heart diseaseand its pathogenesis. Circ Res 1983; 52 (Suppl I): I-147. [4] Yasue H, Touyama M, Tanaka S, Akiyama F. Role of autonomic nervous system in the pathogenesis of Prinzmetal’s variant form of angina pectoris. Circulation 1974; 50: 534-539. [5] Yasue H, Horio Y, Kugiyama K et al. Induction of coronary artery spasm by acetylcholine in patients with variant angina: possible role of the parasympathetic nervous system in the pathogenesis of coronary artery spasm. Circulation 1986; 74: 955-963. [6] Okumura K, Yasue H, Matsuyama K et al. Sensitivity and spcciticity of intracoronary injection of acctylcholine for the induction of coronary artery spasm. J Am Coil Cardiol 1988; 12: 883-888. [7] Okumura K, Yasue H, Kugiyama K et al. Multivessel coronary spasm in patients with variant angina: a study with intracoronary injection of acetylcholine. Circulation 1988;77: 535-542.
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values in variant angina pectoris and fixed coronary artery diseasewith normal subjects. Am J Cardiol 1989; 63: 1441-1445. [32] Yasue H, Takizawa A, Nagao M et al. Long-term prognosis for patients with variant angina and influential factors. Circulation 1988; 78: l-9. [33] Kugiyama K, Yasue H, Okumura K et al. Simultaneous multivessel coronary artery spasm demonstrated by quantitative analysis of thallium-201 single photon emission computed tomography. Am J Cardiol 1987; 60: 1009-1014. [34] Shimokawa H, Tomoike H, Nabeyama S, Yamamoto H, Araki H, Nakamura M. Coronary artery spasm induced in atherosclerotic miniture swine. Science 1983; 221: 560-562. (351 Crea F, Chierchia S, Kaski JC et al. Provocation of coro-
nary spasm by dopamine in patients with active variant angina pectoris. Circulation 1986; 74: 262-269. 1361 Okumura K, Yasue H, Matsuysma K et al. Effect of Hl receptor stimulation on coronary artery diameter in patients with variant angina: comparison with effect of acetylcholine. J Am Co11Cardiol 1991; 17: 338-345. 1371 Matsuyama K, Yasue H, Okumura K et al. Effects of Hl-receptor stimulation on coronary arterial diameter and coronary hemodynamics in humans. Circulation 1990;81: 65-71. [38] Yokoyama M, Akita H, Hirata K et al. Supersensitivity of isolated coronary artery to ergonovine in a patient with variant angina. Am J Med 1990; 89: 507-515.
International
Journal of Cardiology
52 (1995) 223-233
Increased constrictor response to acetylcholine of the isolated coronary arteries from patients with variant angina Kiyotaka Kugiyama *, Toyoaki Murohara, Hirofumi Yasue, Tadashi Kimura, Naritsugu Sakaino, Masamichi Ohgushi, Seigo Sugiyama, Ken Okumura Division of Cardiology, Kumamoto University School of Medicine, Honjo I-1-1, Kumamoto City 860, Japan Received 27 June 1995; revision received 1 August 1995; accepted 31 August 1995
Abstract The aim of this study was to determine whether isolated coronary arteries from patients with variant angina show hyperreactivity and/or supersensitivity to acetylcholine in vitro. Coronary arterial rings were obtained at autopsy within 3 h after death from six coronary arteries having spasmin four patients with variant angina and from 22 coronary arteries in 14 control patients with non-cardiac death. The coronary rings were suspendedin the organ chamber filled with Krebs Henseleit solution bubbled with 95% 0, + 5% CO,, and their isometric tensions were monitered. Arterial rings isolated from both the patients with variant angina and the controls contracted dose-dependently in response to acetylcholine (10-9-10-5 mol/l). EC,, of acetylcholine (i.e. concentration producing 50% of maximum contraction) was not significantly different between the coronary arteries from patients with variant angina and those from controls, but maximum contraction elicited by acetylcholine (expressedas a percentageof the contraction elicited by 60 mmoyl KCl) was significantly greater in the coronary arteries from patients with variant angina than those from controls. In conclusion, the isolated coronary arteries from patients with variant angina have hypercontractile reactivity to acetylcholine. This intrinsic alteration of the coronary reactivity to acetylcholine may play a role in the genesis of coronary spasm occurring in the situations of enhanced parasympathetic nervous discharge. Keywords: Coronary artery spasm;Variant angina; Acetylcholine
1. Introduction It has been shown that coronary artery spasm plays an important role in the wide spectrum of ischemic heart disease [l-3]. Coronary spasm usually occurs at rest, particulary from midnight * Corresponding author, Tel.: +81 96 3442111 (Ext 5872); Fax: +81 96 3623256.
to early morning when the activity of the parasyrnpathetic nervous system is enhanced [3,4]. We have demonstrated that subcutaneous injection of methacholine or intracoronary injection of acetylcholine, a neurotransmitter of the parasympathetic nervous system, induces coronary artery spasm in patients with variant angina, suggesting that parasympathetic nervous system may play an important role in the mechanism(s) and the circadian
0167-5273/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-5273(95)02478-F
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variation of coronary artery spasm [3-81. However, the precise mechanism whereby acetylcholine induces coronary artery spasm in patients with variant angina still remains unknown. Acetylcholine is known to have two distinct effects on vascular tension; one is direct vascular smooth muscle contraction, and the other is endothelium-dependent vasorelaxation [9- 111.Therefore, at least two possible mechanisms of acetylcholine-induced coronary spasmare suggested:(1) a hyperreactivity of the coronary vascular smooth muscle to acetylcholine; (2) an impaired endothelium-dependent vasodilator response to acetylcholine. Furthermore, it also remains to be determined which factors may importantly contribute to the genesis of coronary artery spasm, neurohumoral abnormalities or intrinsic functional alterations of coronary arteries. The in-vivo studies seem to have difficulty in providing conclusive answersfor these questions. Therefore, a study examining vasomotor regulation of the isolated coronary arteries from patients with variant angina is required to determine the mechanism(s) of coronary spasm. Autopsy casesof patients with variant angina are rare, and to our knowledge, there has been no report examining the responseto acetylcholine of the isolated coronary arteries from patients with variant angina. In this study, we examined the effects of acetylcholine on the tonus of isolated coronary arteries from the patients with variant angina in comparison with those from the controls.
cf Cardiology 52 (1995) 223-233 2. Materials and methods
Coronary arterial segmentsat the spastic sites were isolated at autopsy within 3 h after death from six coronary arteries having spasm in four patients with variant angina. The coronary segmentswere also isolated at autopsy within 3 h after death from the mid portion of 22 coronary arteries in 14 patients who had no apparent cardiac diseaseduring their life, serving as controls. 2.1. Patients
A summary of clinical characteristics and cause of death of the patients with variant angina is shown in Table 1. All of the patients had attacks of chest pain associatedwith ST segmentelevation on the electrocardiograms (occurred at rest, usually in the middle of the night or early morning). Coronary artery spasm was angiographically demonstrated in all of the patients during their life at the spontaneous attack or the attack induced by intracoronary injection of acetylcholine [5-81. The angina1 attacks in all of the patients were almost completely suppressedby anti-angina1 drugs which had been discontinued for more than 3 days before death becauseof inability to take orally. All patients with variant angina had no myocardial infaction. Informed consent for cardiac catheterization was obtained from each patient. The causes of death in 14 control patients (52-68 years old, mean age 59, men) were hepato-
Table 1 Clinical characteristics of patients with variant angina Case
Age/Sex Cause of death
ST elevation during attacks
Sites of spasm
Induction of spasm Organic stenosis LAD LCX RCA
1 (F.T.)
64/M
Cholangiocarcinoma
11,111,aVF
2 (K.Y.) 57/M 3 (T.M.) 65/M
Hepatoeellular carcinoma Vt-s Hepatocellular carcinoma II, III, aVF, VI-s
4 (T.V.)
Grawitz’s tumor
52/M
II, III, aVF
RCA, LAD, LAD, RCA, LAD, RCA,
mid proximal proximal proximal proximal mid
ACh (50 pd ACh (50 ml ACh (100 ag) Spontaneous Spontaneous ACh (50 rg)
N
N
25%
N N
N N
N N
75% 75% 90%
Coronary artery spasmwas angiographically demonstrated at the spontaneousattack or the attack induced by intracoronary injection of acetylcholine during their life. Organic stenosis was evaluated after sublingual administration of nitroglycerin. Age, age at death; ST, ST segmentin electrocardiographic leads; LAD, left anterior descendingcoronary artery; LCX, left circumflex coronary artery; RCA, right coronary artery; Mid or proximal, mid or proximal portion of the coronary artery; M, male; N, no organic stenosis.
K. Kugiyama et al. /International
cellular carcinoma (four patients), adenocarcinoma of the stomach (four patients), adenocarcinoma of the colon (four patients), transitional cell cancer of the bladder (one patient), and glioblastoma multiforme (one patient). 2.2. Coronary arteriography in patients with variant angina (Table I) Case 1 (F. T.). The left coronary artery was nor-
mal, and the right coronary artery had a slight nar-
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Journal of Cardiology 52 (1995) 223-233
rowing in the middle position at baseline. Injection of acetylcholine chloride (50 pg; approximately 5 pmol/l equivalent in the left coronary blood circulation) into the left coronary artery induced a subtotal occlusion at the proximal site of the left anterior descending coronary artery, which was associatedwith chest oppression and ST segment depression on the electrocardiographic leads V,-,. The spasmof the left anterior descendingcoronary artery resolved spontaneously. Thereafter, acetyl-
B
.
CASE
1 (RCA1
0
CONTROL
ACETYLCHOLINE.
-log M
Fig. 1. Case 1 (the right coronary artery) in Table 1. Coronary arteriograms showing acetylcholine-induced spasm at cardiac catheterization during their life and in-vitro responses to acetylcholine of the isolated right coronary artery at the site of spasm. (A) The right coronary artery at baseline. (B) Intracoronary injection of acetylcholine (50 pg) induced diffuse spasm (indicated by arrows), which was associated with chest oppression and ST segment elevation on electrocardiographic leads II, III, and aVF. (C) Spasm of the right coronary artery was resolved by intracoronary injection of isosorbide dinitrate. (D) Dose-response curves for acetylcholineinduced contraction of the isolated coronary arteries in vitro. Values in Case 1 are the mean f S.E.M. of the responses to acetylcholine of the four rings prepared from the right coronary artery at the site of the spasm. Values in Control are the mean f S.E.M. of the responses of the 22 rings from the isolated coronary arteries from 14 control patients who had no apparent cardiac disease. Contractions are expressed as a percentage of the contraction elicited by 60 mmovl KCL. -log M = -log [moliI].
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choline chloride (50 pg; approximately 10 pmol/l equivalent in the right coronary blood circulation) was again injected into the right coronary artery and induced a diffuse spasmat the mid position of the right coronary artery, which was associated with chest oppression and ST segmentelevation on electrocardiographic leads II, III, and aVF. The spasmof the right coronary artery was resolved by intracoronary injection of isosorbide dinitrate (Fig. 1). Case2 (K. Y.). The left coronary artery was normal at baseline. Intracoronary injection of acetyl-
Journal of Cardiology 52 (1995) 223-233
choline chloride (100 pg; approximately 10 pmol/l equivalent in the left coronary blood circulation) induced total occlusion at the proximal site of the left anterior descending artery (indicated by an arrow), which was associated with chest oppression and ST segment elevation on electrocardiographic leads Vi+ Spasm of the left anterior descendingcoronary artery was resolved by injection of isosorbide dinitrate into the left coronary artery (Fig. 2). Case3 (TM). Both of the left and right coronary arteries were normal at baseline. Simulta-
Fig. 2. Case 2 in Table 1. (A) Left coronary artery at baseline. (B) Intracoronary injection of acetylcholine (100 pg) induced total occlusion (indicated by an arrow), which was associatedwith chest oppression and ST segmentelevation on electrocardiographic leads VI-,. (C) Spasm of the left coronary artery was resolved by injection of isosorbide dinitrate into the left coronary artery. (D) Doseresponse curves for acetylcholine-induced contraction of the isolated coronary arteries in vitro. Values in Case 2 are the mean * S.E.M. of the responsesto acetylcholine of the four rings prepared from the left anterior descendingcoronary artery at the site of the spasm. Values in Control are same as shown in Fig. 1.
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Journal of Cardiology 52 (1995) 223-233
neous spasm of the right coronary artery (total occlusion) and the left anterior descending coronary artery (subtotal occlusion) occurred spontaneously and was associated with chest oppression and ST segment elevation on the electrocardiographic leads II, III, aVF, and Vim4. A fall in blood pressure and ventricular tachycardia occurred during the spasm.The spasmwas resolved by injection of isosorbide dinitrate into both of the coronary arteries (Figs. 3,4).
227
Case 4 (T. U.). The organic stenoseswere found in the left descending coronary artery (75% stenosis),the left circumflex coronary artery (75% stenosis), and the right coronary artery (90% stenosis). Injection of acetylcholine chloride (50 pg) into the right coronary artery induced a subtotal occlusion just distal to the organic stenosisof the right coronary artery, which was associated with typical substernal chest pain and ST segment elevation on the electrocardiographic leads II, III,
A
6 jz
loo-
.
CASE 3 IRCAl
0
CONTROL
z 5 0
50-
S
Fig. 3. Case3 (the right coronary artery) in Table I. (A) The right coronary artery at baseline. (B) Total occlusion of the right coronary artery (indicated by an arrow) occurred spontaneously, and spasm of the left anterior descending coronary artery also occurred simultaneously. Chest oppression and ST segmentelevation on electrocardiographic leads II, III, aVF, and Vl-, were associated.(C) Spasmof the right coronary artery was resolved by the intracoronary injection of isosorbide dinitrate. (D) Dose-responsecurves for acetylcholine-induced contraction of the isolated right coronary arteries in vitro. Values in Case 3 are the mean f S.E.M. of the responsesto acetylcholine of the four rings from the right coronary artery at the site of the spasm. Values in Control are same as shown in Fig. 1.
228
K. Kugiyama et
al. /International
Journal of Cardiology 52 (1995) 223-233
ACETYLCHOLINE.
-log M
Fig. 4. Case 3 (the left anterior descending coronary artery) in Table 1. Coronary arteriograms during coronary spasm in this case was also described in the legend of Fig. 3. (A) The left coronary artery at baseline. (B) Spasmof the left anterior descendingcoronary artery occurred spontaneously. (C) Spasm was resolved by the intracoronary injection of isosorbide dinitrate. (D) Dose-response curves for acetylcholine-induced contraction of the isolated left anterior descendingcoronary arteries in vitro. Values m Case 3 are the mean f S.E.M. of the responsesto acetylcholine of the four rings from the left anterior descendingcoronary artery at the site of the spasm. Values in Control are same as shown in Fig. 1.
and aVF. The spasmwas resolved by injection of nitroglycerin into the right coronary artery. Coronary angiograms and histological findings of the coronary arteries in Case 4 in Table 1 have been previously reported [ 121. 2.3. Experimental procedure
Coronary segments(20 mm long) at the sites of the spasm in the isolated coronary arteries from patients with variant angina and those at the mid portion of the coronary arteries from the control patients were used for the muscle chamber experi-
ments and the histological examinations. A transverse section of the coronary segmentswas made, and the coronary segmentswere divided into two parts; one part was used for histological examinations and the other part was cleaned from connective tissues and cut into ring segments2 mm long. Each ring was vertically suspended between stainless steel hooks in an organ chamber with 10 ml of Krebs Henseleit solution (in mmol/l; NaCl 118, KC1 4.7, NaH2P04 1.2, MgSO4 1.2, CaCl* 2.0, NaHC03 25, EDTA 0.05, and glucose 10.0). The solution was bubbled with mixed gas (95%
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Journal of Cardiology 52 (1995) 223-233
O2 + 5% CO*) and maintained at 37°C and pH 7.4. The lower hook was anchored to the bottom of organ chamber, while upper one was connected to a force transducer UL30GR (Minebia, Tokyo, Japan), and isometric tension changes were recorded on an ink-writing pen recorder SR6211 (Graphtec, Tokyo, Japan). Rings were progressively stretched over 90 min to the optimum resting tension previously determined to be 4 g. The Krebs Henseleit solution was replaced every 20 min. After the equilibration period, the contractile responseto 60 mmol/l KC1 was examined first, then the rings were washed repeatedly and equilibrated for at least 30 min before the next protocol. The arterial rings that failed to produce > 1 g contraction in response to 60 mmoyl KC1 were considered to be invalid for the vascular smooth musclefunction and therefore were discarded from the study. Thereafter, increasing concentrations of acetylcholine were added cumulatively into the organ chamber. In one or two rings of each artery obtained from the patients with variant angina and the control patients, the increasing concentrations of acetylcholine were added cumulatively after precontraction with prostaglandin Fz, (30 pmol/l) in order to examine endothelium-dependent relaxation to acetylcholine. More than three rings were prepared from one coronary artery and the responseswere averaged and included in the final data analysis. 2.4. Histological examination
The segmentsof the coronary arteries were fixed in 10% formalin solution and embedded in paraffin, then sliced into sections of 3 pm thickness. Histological sections were stained with haematoxylin-eosin and Verhoeff-Van Gieson’s elastic stains. Prostaglandin FZa was obtained from Ono Pharmaceuticals (Osaka, Japan). Other chemicals were from Sigma Chemical (St Louis, MO). Data were expressedas mean f S.E.M. Statistical evaluation of the difference of two meanswas performed by unpaired Student’s t-test. When more than two means were compared, analysis of variance (ANOVA) followed by the Bonferroni’s t-test was used. Differences between values were
considered to be statistically significant when P < 0.05.
The study was in agreementwith the guidelines approved by the ethics committee at our institution. 3. Results
Acetylcholine constricted all coronary arterial rings from controls as well as from patients with variant angina (Fig. l-5). Constrictor responsesto acetylcholine (expressed as a percentage of contraction elicited by 60 mmol/l KCl) are summarized in Fig. 6. Maximal constrictor responses to acetylcholine were significantly greater in the coronary rings from the patients with variant angina than in those from the control patients (138 f 6.2% vs. 92 f 7.2%, n = 6 and 22, respectively, P < 0.01). The ECsOvalue of acetylcholine, the concentration producing 50% of maximum contraction, was not significantly different between the patients with variant angina and the controls (-log [mol/l]; 6.6 f 0.4 and 6.7 f 0.3, n = 6 and 22, respectively). Acetylcholine did not relax any arterial rings tested from both groups after precontraction with prostaglandin Fza. Relaxation induced by sodium nitroprusside (1 pmol/l) was completely preserved in all arterial rings from both groups.
CONTROL
VARIANT ANGlN,i
k;.--
:; ‘: $
\
I Fig. 5. Representative recordingsshowingthe contractile responses to acetylcholine in the isolatedcoronaryarteries from a patientwith variant angina and a control patient. Acetylcholine (ACh) was added to the muscle chamber to give the concentrations shown (expressed as -log [moM]). KCL, the contraction evoked by 60 mmoM KCI; SNP, sodium nitroprnsside (1 PmoyI); W, washing,
230
K. Kugiyama et al. /International 0 VARIANT ANGINA (n=6)
0 CONTROL (n=22) * p
Fig. 6. Dose-responsecurves for acetylcholine-induced contraction of the six isolated coronary arteries having spasm from four patients with variant angina and of the 22 isolated coronary arteries from 14 control patients. Contractions are expressedas a percentageof the contraction elicited by 60 mmohl KCI. -log M = -log [mobs].
3.1. Histological findings
Endothelial cell lining was completely preserved in all arterial sections from the patients with variant angina and the control patients. Intimal hyperplasia was observedin all arterial sections from the patients with variant angina as well as the controls. Intimal calcification and cholesterol crystal deposits were shown in the sections from the left anterior descendingcoronary artery in Case2 and from the right coronary artery in Case 4. No significantly abnormal changewas microscopically observed in media in all arterial sections from the patients with variant angina. There was no apparent abnormal finding in adventitia in the sections from Cases1,2, and 3, but scatteredinfiltration of inflammatory cells and degenerative and fibrotic perivascular autonomic nerves were found in the adventitia of the right coronary artery from Case 4 as reported previously [ 121. 4. Discussion
Coronary artery spasmoccurs most often when patients are at rest, particularly from midnight to early morning, and is usually not provoked by ex-
Journal of Cardiology 52 (1995) 223-233
ercisein the daytime [ 13,141.It is also well known that the activity of the parasympathetic nervous system is enhanced at rest and is suppressed by physical activity [ 15,161. Furthermore, we have shown that subcutaneous injection of methacholine or intracoronary injection of acetylcholine, the neurotransmitter of the parasympathetic nervous system, induces coronary artery spasm and that the attack can be suppressed by atropine, a parasympatholytic agent, in some patients with variant angina [4-71. We therefore postulated that the activity of the parasympathetic nervous system might be related to the pathogenesisand circadian variation of variant angina or coronary spasm. The present study demonstrated that the isolated coronary arteries from the patients with variant angina had greater constriction than those from the controls in response to acetylcholine at the concentrations of l-10 pmol/l, and these concentrations were the same as those infused into the coronary artery in vivo human for the induction of coronary artery spasm at cardiac catheterization [5-71. The present result indicates that the increased contractile responseof the coronary arteries to acetylcholine in patients with variant angina seems not to be caused by the neurohumoral factors but is intrinsic to the coronary arteries in these patients. This hyperreactivity to acetylcholine in the coronary arteries could play a role in the genesisof coronary spasm occurring in the condition of enhanced parasympathetic nervous discharge. This is in agreement with the phenomenon observed in the swine model of coronary spasm 1171.
Coronary artery spasm is angiographically defined as a total occlusion or a subtotal occlusion of the epicardial arteries leading to myocardial ischemia [l-7] and should be distinguished from the non-occlusive vasoconstriction observed in the atherosclerotic coronary arteries in humans [ 18-201 or experimentally endothelium-denuded arteries in animal models [lo]. The atherosclerotic coronary arteries might exhibit supersensitivity (samedegreeof diameter reduction as that in normal arteries occurs in responseto a lower dose of constrictor agonists), but the maximal reduction of arterial diameter is usually ~30% and is not enough to cause myocardial ischemia [6,18-211.
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Journal of Cardiology 52 (1995) 223-233
On the other hand, coronary arteries involved in spasm do not necessarily have hypersensitivity in response to the intracoronary infusion of the agonists but exhibit increased maximal contractile response (hyperreactivity) sufficiently to occlude arteries in angiographic studies [22-241. From this point of view, the present study shows that the isolated coronary arteries from patients with variant angina exhibit hyperreactivity (expressedas a maximal contraction) but not hypersensitivity (expressed as EC,,) in response to acetylcholine, which may be in agreementwith the characteristic contractile responsesof coronary arteries having spasm to intracoronary infusion of acetylcholine, observed in angiographic studies. The balance between two opposing muscarinic effects, endothelium-mediated vasorelaxation and direct stimulation of smooth muscle, determines vasomoter responses to acetylcholine [9- 1I]. Acetylcholine has been shown to cause endothelium-dependent relaxation in normal vessels of most spieces [9,11,25]. However, relaxation to acetylcholine was not shown in any coronary arteries tested from the patients with variant angina as well as the control patients in the present study, although histological examination showed complete preservation of endothelial lining of the coronary arteries from both groups. There are conflicting results in the muscarinic endotheliumdependent relaxation of the isolated human coronary arteries. Ginsburg [26] and other workers [27] reported that isolated human coronary arteries obtained from hearts of transplant recipients or cadavers lack the phenomenon of endothelium-dependent relaxation and constrict in response to acetylcholine. Bossaller et al. [25] however, showed that non-atherosclerotic coronary arteries from the hearts of transplant recipients exhibit muscarinic endothelium-dependent relaxation. It is now well-known that atherosclerosis impairs muscarinic endothelium-dependent relaxation [19-20,251 and begins in childhood and progresseswith increasing age [28,29]. In the present study, intimal thickening was present in the coronary arteries isolated from the patients with variant angina as well as those from the controls, which may cause defective muscarinic endothelium-dependent relaxation.
231
The observed hypercontractile responses to acetylcholine in coronary arteries from the patients with variant angina seem to have resulted from the enhanced responseof the arterial smooth muscle to acetylcholine. The precise mechanism for the hyperreactive contractility, without change of EC!sOvalues in response to acetylcholine, remains entirely unknown. The change of the receptor population and/or the post-receptor signalling pathway linked to the contractile protein in smooth muscle might be involved in the mechanism explaining for the present findings. Clinical studies [ 18-201 and in-vitro experiments [21,25,30] show that hypercholesterolemia and atherosclerosis enhance constrictor responses of arterial smooth muscle to various agonists. The present pathological examination showed substantial intimal thickening in coronary rings from patients with variant angina. Therefore, atherosclerosis may partly play a role in the mechanisms of the hypercontractile responseto acetylcholine in coronary arteries from patients with variant angina. However, hyperlipidemia is not a risk factor in patients with variant angina [31,32] and we have found that spasmoccurs often in angiographically normal coronary arteries, especially in patients with multivessel coronary artery spasm [7,33]. Therefore, other unknown mechanism(s)as well as atherosclerosis could contribute to the hypercontractility of the coronary arterial smooth muscle in response to acetylcholine in patients with variant angina. Coronary artery spasmcan be induced in patients with variant angina or miniture swine by various agonists such as ergonovine, dopamine, and histamine as well as acetylcholine [5,17,22,23,35-371.It remains undetermined in the present study whether the hyperreactive contractile responseof the isolated coronary arteries from patients with variant angina may be also shown to these agonists known to provoke coronary spasm. Yokoyama et al. [38] reported a case of variant angina whose post-mortem isolated coronary artery showed supersensitivity to ergonovine. Although they did not address the hyperreactivity against hypersensitivity, their data showed hyperreactivity to ergonovine of the isolated coronary artery from patients with variant angina. The enhanced responsivenessof the coronary artery to
232
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agonists with disparate surface receptors in patients with variant angina suggestsfunctional alteration(s) of the post-receptor regulatory pathway, shared by the different receptor stimulations, for smooth muscle contraction [24]. In conclusion, the isolated coronary arteries from patients with variant angina have the increased reactivity to acetylcholine. The hyperreactivity of the coronary arteries in response to acetylcholine could contribute to the coronary artery spasm occurring at rest when the activity of the parasympathetic nervous system is enhanced, supporting the hypothesis that parasympathetic nervous system may play an important role in the pathogenesis and the circadian variation of the attack of variant angina or coronary artery spasm. Acknowledgement This study was supported in part by grants-inaid for Scientific Research on Priority Area (03268107), B03454257, and C3670460 from the Ministry of Education, Science, and Culture in Japan and the Smoking Research Foundation, Tokyo, Japan. References [1] Hills LD, Braunwald E. Coronary artery spasm. N Engl J Med 1978;299: 695-702. [2] Maseri A, Severi S, DeNes M et al. Variant angina: one aspect of a continuous spectrum of vasospastic myocardial ischemia. Am J Cariol 1978;42: 1019-1035. [3] Yasue H, Omote S, Takizawa A, Nagao M. Coronary arterial spasmin ischemic heart diseaseand its pathogenesis. Circ Res 1983; 52 (Suppl I): I-147. [4] Yasue H, Touyama M, Tanaka S, Akiyama F. Role of autonomic nervous system in the pathogenesis of Prinzmetal’s variant form of angina pectoris. Circulation 1974; 50: 534-539. [5] Yasue H, Horio Y, Kugiyama K et al. Induction of coronary artery spasm by acetylcholine in patients with variant angina: possible role of the parasympathetic nervous system in the pathogenesis of coronary artery spasm. Circulation 1986; 74: 955-963. [6] Okumura K, Yasue H, Matsuyama K et al. Sensitivity and spcciticity of intracoronary injection of acctylcholine for the induction of coronary artery spasm. J Am Coil Cardiol 1988; 12: 883-888. [7] Okumura K, Yasue H, Kugiyama K et al. Multivessel coronary spasm in patients with variant angina: a study with intracoronary injection of acetylcholine. Circulation 1988;77: 535-542.
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[al Yasue H, Touyama M, Shimamoto M, Kato H, Tanaka S, Akiyama F. Role of autonomic nervous system in the pathogenesisof Prinzmetal’s variant form of angina pectot%. Circulation 1974;50: 534-539. [91 Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288: 373-376. Vu1 Vanhoutte PM. The endothelium-modulator of vascular smooth muscle tone. N Engl J Med 1988;319: 512-513. 1111Ignarro LJ. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res 1989; 65: l-21. 1121Jougasaki M, Yasue H, Takahashi K. Perivascular nerve lesion of the coronary artery involved in spasm in a patient with variant angina. Pathology 1989;21: 304-307. 1131 Prinzmetal M, Kennamer R, Meliss R, Wada T, Bor T. Angina pectoris. I. A variant form of angina pectoris. Am J Med 1959;27: 357-388. u41 Yasue H, Omote S, Takizawa A, Nagao M, Miwa K, Tanaka S. Circadian variation of exercisecapacity in patients with Prinxmetal’s variant angina: role of exerciseinduced coronary arterial spasm. Circulation 1979; 59: 938-948. 1151 Robinson BF, Epstein SE, Beiser GD, Braunwald E. Control of heart rate by the autonomic nervous system. Studies in man on the interaction between baroreceptor mechanismsand exercise. Circ Res 1966; 19: 400-408. [If4 Vatner SF, Pagani M. Cardiovascular adjustments to exercise:hemodynamicsand mechanisms.Prog Cardiovasc Dis 1976; 19: 91-103. ]171 Fukai T, Egashira K, Hata H et al. Serotonin-induced coronary spasm in a swine model, a minor role of defective endothelium-derived relaxing factor. Circulation 1993;88: 1922-1930. 1181Yasue H, Matsuyama K, Matsuyama K, Okumura K, Morikami Y, Ogawa H. Response of angiographically normal human coronary arteries to intracoronary injection of acetylcholine by age and segment:possible role of early coronary atherosclerosis. Circulation 1990; 81: 482-490. [I91 Vita JA, Treasure CB, Yeung AC et al. Patients with evi-
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