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Cardiovascular Research 1999 41(1):267-274; doi:10.1016/S0008-6363(98)00162-X
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Copyright © 1999, European Society of Cardiology

Identification of mRNA for 5-HT1 and 5-HT2 receptor subtypes in human coronary arteries

Tatsuro Ishida, Ken-ichi Hirata, Tsuyoshi Sakoda, Seinosuke Kawashima, Hozuka Akita and Mitsuhiro Yokoyama*

The First Department of Internal Medicine, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

* Corresponding author. Tel.: +81-78-341-7451, ext. 5500; fax: +81-78-341-1390; e-mail: yokoyama@med.kobe-u.ac.jp

Received 16 January 1998; accepted 30 April 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Although pharmacological studies have indicated that serotonin (5-HT)-evoked contraction of the human coronary artery is mediated by 5-HT1-like and 5-HT2 receptors, the gene expression of 5-HT receptors is still unclear. We examined mRNA expression of 5-HT1 and 5-HT2 receptor subtypes in human coronary arteries. Methods: Total RNA was extracted from human coronary arteries of 14 patients at autopsy by the guanidine method. Reverse transcription-polymerase chain reaction (RT–PCR) and ribonuclease protection assays were performed to identify 5-HT1 and 5-HT2 receptor mRNA expression in human coronary artery. Results: By RT–PCR, 5-HT1B, 5-HT2A and 5-HT2B mRNAs were detected in all of the 14 patients. 5-HT1A, 5-HT1D, and 5-HT1E mRNAs were detected in only some patients. However, neither 5-HT1F mRNA nor 5-HT2C mRNA was detected in any patient. By ribonuclease protection assay, 5-HT1B and 5-HT2A signals were detected in all patients examined, but neither 5-HT1A, 5-HT1D nor 5-HT2B signal was detected in any patient. Conclusions: Of 5-HT1/2 receptor subtypes, 5-HT1B and 5-HT2A receptor mRNAs were predominantly expressed in human coronary arteries. Our finding provides molecular evidence that the 5-HT1B receptor may be the 5-HT1-like receptor which mediates constriction of human coronary arteries.

KEYWORDS Human coronary artery; Serotonin; 5-HT1B receptor; 5-HT2A receptor; Ribonuclease protection assay


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Serotonin (5-hydroxytryptamine; 5-HT), which is released from activated platelets, is a potent vasoactive and platelet-aggregating substance [1–4]. Plasma serotonin levels in coronary sinus have been shown to be increased in patients with limiting angina and coronary lesions [3, 4], and extensive evidence has shown that serotonin plays an important role in the pathogenesis of ischemic heart diseases including acute coronary syndrome [1–5]and coronary spasm [6–8]. Ergonovine is the most commonly used and powerful agent for provocation of coronary spasm, which is mediated mainly by 5-HT receptors [9–11]. Recently, augmented arterial contractile responses to serotonin and to ergonovine have been demonstrated in clinical investigations of patients with coronary atherosclerosis [12, 13]or variant angina [13–17]. The augmented contractile response to serotonin is also demonstrated in animal models of atherosclerosis such as high cholesterol diet-fed animals [18]and hereditary hyperlipidemic rabbits [19].

In pharmacological characterization of receptor subtypes [20]using 5-HT receptor agonists and antagonists, serotonin has been found to contract human large coronary arteries through 5-HT1-like and 5-HT2 receptors [21–24]. However, at present there are limitations to pharmacological characterization of 5-HT receptor subtypes in the vasculature, since agonists, antagonists and antibodies sufficient to distinguish all 5-HT receptor subtypes are not yet available, and little is known concerning the gene expression of 5-HT receptors in coronary vessels. We therefore employed reverse transcription-polymerase chain reaction (RT–PCR) and ribonuclease protection assays to identify the gene expression of 5-HT1 and 5-HT2 receptor subtypes in human coronary arteries. We found that human coronary arteries express both 5-HT1 and 5-HT2 receptor mRNAs, and that, in particular, 5-HT1B (previously referred to as 5-HT1Dβ [25]) and 5-HT2A receptor genes are predominantly expressed in human coronary arteries.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Patients
This study conformed with the principles outlined in the Declaration of Helsinki (Br Med J 1964;ii:177). It was performed with approval of the Institutional Review Board on human research at Kobe University. Human coronary arteries were obtained 2–6 h after death (average 2.5±0.6 h) during autopsy from 14 patients (mean age 64.1±14.3 years, ten males and four females) with a variety of clinical conditions. Age, sex, and pathological diagnoses of the patients are outlined in Table 1. None of the patients had a documented history of ischemic heart disease.


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  		Table 1 The characteristics of the patients. The age, sex, pathological diagnoses, and results of RT–PCR analysis

 
2.2 Preparation of total RNA from human coronary arteries
Both right and left large epicardial coronary arteries were dissected from the heart and washed with phosphate-buffered saline (PBS). The adventitia was carefully removed and the endothelium was denuded by rubbing the vessels with filter paper moistened with PBS. Each coronary sample was frozen in liquid nitrogen and then transported to the laboratory. Total RNA was immediately extracted by the acid guanidinium thiocyanate–phenol–chloroform method of Chomczynski and Sacchi [26]with modifications to be described. Briefly, coronary artery tissues were homogenized with a Polytron homogenizer (Kinematica) in 20 ml of lysis solution (4 mol/l guanidinium thiocyanate, 25 mmol/l sodium citrate, 0.5% lauryl sarcosin). After water-saturated phenol–chloroform extraction, they were precipitated by adding 2-propanol and centrifuged at 15 000g for 30 min. The RNA pellets were resuspended in 3 ml of the lysis solution. After repeated Tris-saturated phenol–chloroform extraction, they were precipitated by adding ethanol and stored at –80°C until use. At the time of study, the RNA samples were centrifuged at 15 000g for 15 min and resuspended in ribonuclease-free water.

2.3 RT–PCR
Total RNA (5 µg) was incubated at 37°C for 2 h with oligo(dT)15 primers (Promega) and M-MLV reverse transcriptase (GIBCO BRL) in the reaction buffer to synthesize single-stranded cDNA. The reaction buffer contained the following: 50 mmol/l Tris–HCl (pH 8.3), 75 mmol/l KCl, 10 mmol/l dithiothreitol, and 3 mmol/l MgCl2. Based on the published sequences of human 5-HT receptors [27–33], we designed PCR primers which distinguish all known 5-HT1 and 5-HT2 receptors. Human glyceraldehyde-3-phosphate dehydrogenase [34](GAPDH) was used as a control gene in PCR analysis. Forward and reverse primers for each 5-HT receptor and GAPDH are shown in Table 2 (top). Double-stranded cDNAs were synthesized and amplified using 1 U Taq polymerase (TAKARA) and 80 nmol/l primers in 0.05 ml of 10 mmol/l Tris–HCl buffer (pH 8.3), 50 mmol/l KCl, 2.0 mmol/l MgCl2, 0.5 mmol/l dNTP, 2 mmol/l dithiothreitol, and 0.01% gelatin for 35 cycles at 93°C, 54°C, and 72°C for 1, 2 and 3 min, respectively. The PCR products were checked with 0.75% agarose gel electrophoresis and then identified by sequencing with the dideoxy method (Sequenase Version II DNA sequencing kit, USB) or by endonuclease digestion pattern.


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  		Table 2 Top: Oligonucleotide primers used in RT–PCR analysis. Bottom: Oligonucleotide primers used to synthesize 5-HT receptor cDNAs in ribonuclease protection assay

 
2.4 Ribonuclease protection assay
To evaluate the expression of 5-HT receptor mRNA, we performed ribonuclease protection assays. Based on the reported sequences of the 5-HT1A/1B/1D/1E/2A/2B receptors [27–30, 32, 33], 32P-labeled riboprobes which specifically recognized mRNA encoding the first extracellular domain of each 5-HT receptor were generated. The regions of human 5-HT receptor cDNAs (from +1 to +308 of 5-HT1A, from +1 to +214 of 5-HT1B, from +1 to +240 of 5-HT1D, from +1 to +173 of 5-HT1E, from +1 to +239 of 5-HT2A, and from +1 to +188 of 5-HT2B receptors) were amplified with the forward and reverse primers shown in Table 2 (bottom). The templates used in PCR reaction were human genomic DNAs extracted from whole blood (for 5-HT1 receptors) or human coronary artery cDNAs (for 5-HT2 receptors). The amplified DNAs of 5-HT1A/1B/1D/2A and 5-HT1E/2B receptors were subcloned into the pGEM-4Z plasmid (Promega) and the pCR II plasmid (Invitrogen), respectively. These constructed plasmids were sequenced by the dideoxy method and identified. To prepare radiolabeled antisense riboprobes, these plasmids were linearized with adequate endonuclease and transcribed with T7 (for 5-HT1A/1D/1E receptors) or SP6 (for 5-HT1B/2A/2B receptors) RNA polymerase (Promega). Ribonuclease protection assay was performed using a PRAII kit (Ambion). Briefly, an aliquot of total RNA (30 µg) was hybridized with 32P-labeled riboprobes for 14–16 h at 50°C in 80% formamide hybridization buffer, followed by digestion with ribonuclease A and ribonuclease T1 at 37°C for 30 min. After simultaneous precipitation of RNA and inactivation of ribonuclease, the protected fragments were separated in 8 mol/l urea–5% acrylamide denaturing gel by electrophoresis and then exposed to an Imaging Plate (BASIII, FUJI XEROX). The relative signal intensities were determined with an Autoimage Analyzer (BAS2000, FUJI XEROX) [35].


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Analysis of 5-HT receptor subtypes by RT–PCR
To detect low levels of 5-HT receptor mRNA, we performed RT–PCR using primers specific for each of the 5-HT1 and 5-HT2 receptor subtypes. Contamination with genomic DNA was excluded in all RNA preparations by performing the same RT–PCR procedure without addition of reverse transcriptase. GAPDH mRNA, used as a control gene, was detected in all coronary preparations, suggesting that effects of degradation of RNA samples could be excluded or were negligible. After RT–PCR amplification of the cDNA from human coronary arteries, we obtained 1.1–1.4 kbp PCR products which were consistent with the 5-HT1A, 5-HT1B, 5-HT1D (previously referred to as 5-HT1D{alpha} [25]), 5-HT1E, 5-HT2A and 5-HT2B receptors. Typical results of agarose gel electrophoresis for one patient are shown in Fig. 1. The PCR products were confirmed to be each of the 5-HT receptor subtypes by sequencing with the dideoxy method or by endonuclease digestion. Of the 5-HT1 receptor subtypes, 5-HT1B and 5-HT1D receptor mRNAs were detected in 100% and 93% of the studied patients, respectively, but 5-HT1A and 5-HT1E receptor mRNAs were detected in only 50% and 36% of the patients, respectively. There was no relationship between expression of 5-HT1A, 5-HT1D, or 5-HT1E receptor mRNA and patient clinical profile, i.e. age, gender, and cause of death. Of the 5-HT2 receptor subtypes, 5-HT2A and 5-HT2B receptor mRNAs were amplified in all of the patients. On the other hand, neither 5-HT1F nor 5-HT2C (previously referred to as 5-HT1C) receptor mRNA was detected in any of the patients examined. Both 5-HT1F and 5-HT2C receptor genes were amplified by RT–PCR when human brain cDNA was used as a template (data not shown). The results of RT–PCR for the 14 patients are shown in Table 1.


Figure 1
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  		Fig. 1 Typical results of agarose gel electrophoresis of PCR-amplified products derived from cDNA of the coronary artery of a patient (No. 5). As a control for the absence of DNA contamination, all RT–PCRs were performed with or without the addition of reverse transcriptase (+ or – RT) to the cDNA synthesis reaction. Lane 1, 18: {lambda} DNA/HindIII digests, lanes 2, 3: 5-HT1A (1266 bp), lanes 4, 5: 5-HT1D (1134 bp), lanes 6, 7: 5-HT1B (1173 bp), lanes 8, 9: 5-HT1E (1098 bp), lanes 10, 11: 5-HT1F (1101 bp), lanes 12, 13: 5-HT2A (1416 bp), lanes 14, 15: 5-HT2B (1446 bp), lanes 16, 17: 5-HT2C (1377 bp) receptors, lanes 19, 20: GAPDH (1151 bp). The sizes of the putative PCR products are shown in parentheses.

 
3.2 Analysis of 5-HT receptor subtypes by ribonuclease protection assay
To determine the levels of expression of 5-HT receptor mRNAs detected with RT–PCR, we performed ribonuclease protection assays using 32P-labeled riboprobes for 5-HT1A/1B/1D/1E/2A/2B receptors. We did not perform this assay for 5-HT1F/2C receptors, since 5-HT1F/2C mRNAs were not detected by RT–PCR, which provides superior sensitivity. Typical results are shown in Fig. 2.


Figure 2
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  		Fig. 2 Typical results of ribonuclease protection assay using total RNA (30 µg) derived from human coronary arteries of two patients. A: For 5-HT1 receptors, the following riboprobes were used (full length, and protected size of the riboprobes in parentheses, respectively): 5-HT1A (338, 308 bases), 5-HT1B (272, 214 bases), 5-HT1D (276, 240 bases), 5-HT1E (318, 173 bases). Lanes 1–4: full-length riboprobes for 5-HT1A (lane 1), 5-HT1B (lane 2), 5-HT1D (lane 3), 5-HT1E (lane 4) receptors. Lanes 5, 6: Cohybridization of human coronary artery RNA with the 5-HT1A/1B/1D/1E riboprobes. Lane 7: Cohybridization of human total brain RNA with the 5-HT1A/1B/1D/1E riboprobes as positive control for 5-HT1A/1B/1D/1E receptor mRNAs. The size markers refer to lanes 5–7. B: For 5-HT2 receptors, the following riboprobes were used: 5-HT2A (297, 239 bases), 5-HT2B (299, 188 bases). Lanes 1, 2: full-length probes for 5-HT2A (lane 1), 5-HT2B (lane 2) receptors, Lanes 3, 4: Cohybridization of human coronary artery RNA with 5-HT2A/2B riboprobes. Lane 5: Hybridization of human brain RNA with the 5-HT2A riboprobes as positive control for 5-HT2A receptor mRNA. Lane 6: Hybridization of human small intestine total RNA with the 5-HT2B riboprobes as positive control for 5-HT2B receptor mRNA. The size markers refer to lanes 3–6.

 
Probes for 5-HT1A/1B/1D/1E receptors were confirmed to hybridize correctly with positive control RNA. Cohybridization of the 5-HT1A/1B/1D/1E receptors’ riboprobes with human brain total RNA, followed by digestion with single strand-specific ribonucleases and denaturing polyacrylamide gel electrophoresis, yielded protected riboprobe fragments of 308, 214, 240 and 173 bases in length, consistent with 5-HT1A, 5-HT1B, 5-HT1D and 5-HT1E receptors, respectively (Fig. 2A, lane 7). Cohybridization of these riboprobes with total RNA extracted from human coronary arteries revealed the presence of the protected riboprobe fragments of 214 bases in length, consistent with the 5-HT1B receptor. Of the four 5-HT1 receptor subtypes amplified by RT–PCR, only the 5-HT1B receptor mRNA was detected by ribonuclease protection assay in all patients examined (Fig. 2A, lane 5 and 6). Neither 5-HT1A, 5-HT1D nor 5-HT1E receptor mRNA was detected in any patient. The ribonuclease protection assays revealed that levels of mRNA for 5-HT1B receptor were higher than those for other 5-HT1 receptor subtypes in human coronary arteries.

Probes for 5-HT2A/2B receptors were also confirmed to hybridize with positive control RNA. Hybridization of the 5-HT2A receptor riboprobe with human brain total RNA, yielded the protected riboprobe fragment of 239 bases in length, consistent with the 5-HT2A receptor (Fig. 2B, lane 5), and hybridization of the 5-HT2B receptor riboprobe with human small intestine total RNA demonstrated the protected riboprobe fragment of 188 bases in length, consistent with the 5-HT2B receptor (Fig. 2B, lane 6). Cohybridization of the 5-HT2A and 5-HT2B receptor riboprobes with total RNA extracted from human coronary arteries showed the presence of the protected riboprobe fragments of 239 bases in length, consistent with 5-HT2A receptor (Fig. 2B, lanes 3 and 4). 5-HT2A receptor mRNA was detected in all patients, but 5-HT2B receptor mRNA was not detected in any patient. The results of RT–PCR and ribonuclease protection assays are summarized in Table 3.


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  		Table 3 Summary of the results of RT–PCR and ribonuclease protection assay

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In the present study, we identified the subtypes of coronary artery 5-HT receptors using total RNA isolated from human coronary artery tissues. RT–PCR and ribonuclease protection assay revealed that 5-HT1B, and 5-HT2A receptor mRNAs were expressed in all human coronary arteries examined. The detection of 5-HT1B receptor mRNA is consistent with the findings of our own previous PCR studies [36]and that of Bouchelet et al. [37]. 5-HT1A, 5-HT1D, 5-HT1E and 5-HT2B receptor mRNAs were detected only by RT–PCR in some coronary preparations, but not at all by ribonuclease protection assays. We found no relationship between patient clinical profile and expression of 5-HT1A, 5-HT1D, or 5-HT1E receptor mRNA in the RT–PCR study. The inconsistent results of the PCR study remain unexplained, but are probably related to the differences among expression levels of these receptor mRNAs in coronary tissues. No signals were found in the ribonuclease protection assay that did not appear in RT–PCR, suggesting that RT–PCR has higher sensitivity, although it does not yield quantitative results. Our findings suggest that the levels of expression of 5-HT1B and 5-HT2A receptor mRNAs are much higher than those of 5-HT1A, 5-HT1D, 5-HT1E and 5-HT2B receptor mRNAs in human coronary arteries. It may be thus concluded that 5-HT1B and 5-HT2A receptor mRNAs are predominantly expressed among the several 5-HT1 and 5-HT2 receptor subtypes in human coronary arteries, respectively.

The expression of 5-HT1A/1D/1E/2B receptor mRNA was detected in the present PCR study, but the origin of these mRNAs remains unclear. Ullmer et al. [38]demonstrated low expression of 5-HT2B receptor mRNA in cultured human aortic and pulmonary arterial smooth muscle cells by RT–PCR. Indeed, there are some reports that 5-HT2B receptor mediates smooth muscle contraction in rat mesenteric artery [39]. On the other hand, Hamel et al. found expression of 5-HT1D receptor mRNA in some of human cerebral arterial preparations by RT–PCR [40]. Interestingly, they failed to detect this expression by Northern blot hybridization [41], while 5-HT1B receptor mRNA was detected by both RT–PCR and Northern blot hybridization [40, 41]. They speculated that this discrepancy in findings was related to levels of mRNA for the receptor, and that 5-HT1D receptors are expressed at very low levels in some cerebrovascular preparations [40]. Similarly, our finding that expression of 5-HT1A/1D/1E/2B receptor mRNAs in some coronary arteries was detectable only by RT–PCR may also reflect the very low and/or rare expression of these receptors in coronary vessels, although previous reports have excluded the involvement of these receptors in serotonin-evoked vasoconstriction of human coronary arteries [21, 22].

Another possible explanation for the inconsistent findings of PCR is contamination by various minor cell components. Since both the endothelium and adventitia were removed carefully in the present study, the major cell population in the coronary artery specimens studied was thought to be vascular smooth muscle cells. However, we cannot entirely exclude contamination by other minor cell components such as endothelial cells, fibroblasts, macrophages or autonomic nerves, since our study was performed with whole tissues. In particular, in light of the finding of a recent study [38]of expression of 5-HT2B receptor mRNA in cultured human coronary endothelial cells, it seems possible that the 5-HT2B receptor mRNA in our PCR study might have been derived from a small amount of undenuded endothelial cells. Similarly, there is a report suggesting low expression of 5-HT1D receptor in vascular endothelial cells [42]. 5-HT1A receptor mRNA has been reported to be expressed in a variety of tissues and cells in which the localization and function of the 5-HT1A receptor are unclear [27, 43]. On the other hand, 5-HT1E mRNA has not been found in peripheral tissues. The localization and function of these receptor subtypes in coronary tissues remain unclear and should be clarified. However, the detection of 5-HT1B and 5-HT2A receptors by ribonuclease protection assay in all of our patients suggests that these two receptor mRNAs are expressed to a significant extent in human coronary arteries.

It has been demonstrated that two subtypes of 5-HT receptors, 5-HT1-like and 5-HT2 receptors, mediate serotonin-evoked coronary artery contraction [21–24]. A recent study [22]by Kaumann et al. indicated that the 5-HT1-like receptor pharmacologically resembles the 5-HT1B receptor and predominates over 5-HT2 receptors in mediating serotonin-induced contractions. Our detection of 5-HT1B/2A receptor mRNAs in all coronary arteries examined is consistent with the results of pharmacological studies and strongly support their suggestion that the 5-HT1-like receptor is the 5-HT1B in type. On the contrary, 5-HT1A/1D/1E/2B receptors do not appear to participate in serotonin-evoked vasoconstriction of human coronary arteries [21, 22]. Thus, it might be concluded that serotonin-induced coronary artery contraction is evoked as the net effect of activation of these receptors. On the other hand, it is interesting that serotonin and thromboxane A2 (TXA2) have been reported to act synergistically in stimulating vasoconstriction [44–46], and that 5-HT1B receptor-mediated vasoconstriction is preferentially augmented by TXA2 in vasospastic diseases. Future studies of the interaction of TXA2 with these 5-HT receptor subtypes may identify a new mechanism of serotonin-induced vasoconstriction.

Some studies have suggested the presence of an altered 5-HT receptor profile in diseased human coronary arteries. Chester et al. [47]reported that both 5-HT1-like and 5-HT2 receptors mediate contraction of human coronary arteries, and that the effects mediated by 5-HT1-like receptors but not those mediated by 5-HT2 receptors were preserved in patients with ischemic heart disease. Their study suggests that 5-HT1-like receptors predominate over 5-HT2 receptors in atherosclerotic human coronary arteries. In addition, the serotonin-induced coronary contraction was not completely eliminated by ketanserin, a selective 5-HT2A receptor antagonist, in patients with stable angina [16]or variant angina [16, 48]. However, Kaumann and colleagues demonstrated that the response to sumatriptan (a 5-HT1B/1D receptor-agonist) in human coronary arteries exhibited a high degree of variability, which was not directly related to the underlying disease, patient age or degree of atherosclerosis [22]. Although the relative contribution of 5-HT1-like and 5-HT2 receptors in vasoconstriction may vary largely among patients, the enhanced responsiveness to serotonin is observed in at least some coronary arteries with atherosclerosis [14, 15, 47]. Thus, the issue of augmented serotonin-evoked vasoconstriction might not be irrelevant to the finding of altered 5-HT receptor mRNA levels. Moreover, levels of 5-HT1B mRNA, but not of 5-HT2A mRNA, were found to be increased in atherosclerotic lesions in our preliminary study [49]. Further studies must be performed to verify the alterations in 5-HT receptor mRNA levels in atherosclerotic coronary arteries.

In the present study, we examined the expression of mRNAs for only 5-HT1 and 5-HT2 receptor subtypes, since previous pharmacological studies indicated that 5-HT1/2 receptors are principally responsible for the serotonin-induced contraction of human coronary arteries. On the other hand, it should be noted that the recently-cloned 5-HT7 receptor [50], which activates adenylate cyclase, is expressed in human coronary arteries and uterine artery smooth muscle cells [51]. Accumulating evidence demonstrates that the 5-HT7 receptor mediates the serotonin-induced endothelium-independent dilatation/relaxation of a variety of blood vessels [52–54]and the long-lasting hypotensive effects of serotonin [55]. It has thus been postulated that 5-HT7 receptors modulate the vasoconstrictive effects of 5-HT1B or 5-HT2A receptor activation [38, 56].

In conclusion, we analyzed the gene expression of 5-HT1 and 5-HT2 receptor subtypes in human coronary arteries, and found that all coronary arteries examined exhibited expression of mRNAs for 5-HT1B and 5-HT2A receptors. Our findings support previous suggestions that the 5-HT1B receptor is the 5-HT1-like receptor that mediates constriction of human coronary arteries. We therefore speculate that 5-HT1B receptor antagonists, if clinically available, would be useful in treating ischemic heart diseases including variant angina.

Time for primary review 20 days


   Acknowledgements
 
The investigation was supported by Grant-in-Aid for Scientific Research (A2) [No.07557345, 1995-1996] from the Ministry of Education, Science, Sports and Culture, Japan. We are grateful to Dr. Tetsuya Nakamura, Dr. Toshio Okada, Dr. Tetsuro Mizutani (Kobe National Hospital), Dr. Makoto Hatani (Rokko Hospital) and Dr. Kazuo Ohnishi (Kobe Rosai Hospital) for providing human coronary arteries and Seiko Tsutsui for her skillful technical help.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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