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(Circulation. 2004;110:2708-2712.)
© 2004 American Heart Association, Inc.
Vascular Medicine |
From the Department of Medical Physiology, Cardiovascular Research Institute, College of Medicine, The Texas A&M University System Health Science Center, College Station, Tex.
Correspondence to Lih Kuo, PhD, Department of Medical Physiology, Cardiovascular Research Institute, College of Medicine, The Texas A&M University System Health Science Center, 702 Southwest H.K. Dodgen Loop, Temple, TX 76504. E-mail LKUO{at}tamu.edu
Received December 16, 2003; de novo received April 6, 2004; revision received May 24, 2004; accepted May 25, 2004.
| Abstract |
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Methods and Results Porcine subepicardial and subendocardial arterioles (<100 µm) were isolated from the left ventricle and pressurized for in vitro study of vasodilation to the nonselective ß-adrenoceptor agonist isoproterenol and the selective ß2-adrenoceptor agonist procaterol. Both vessel types developed a similar level of basal tone and dilated to isoproterenol and procaterol. However, subepicardial arterioles exhibited a much higher sensitivity and greater dilation capacity to both agonists. The isoproterenol-induced vasodilations were inhibited by glibenclamide, an ATP-sensitive potassium (KATP) channel blocker. In contrast to isoproterenol, dilations of subepicardial and subendocardial arterioles to pinacidil, a KATP channel opener, were similar. In both vessel types, isoproterenol-induced dilation was inhibited by the ß2-adrenoceptor blocker ICI-118,551 but was insensitive to the ß1-adrenoceptor blocker atenolol. Reverse transcriptionpolymerase chain reaction and immunohistochemical data revealed that ß2-adrenoceptor mRNA and protein expression, respectively, were markedly greater in subepicardial arterioles.
Conclusions This study demonstrates that selective activation of ß2-adrenoceptors elicits dilation of both subepicardial and subendocardial arterioles through opening of KATP channels. The higher ß2-adrenoceptor expression in subepicardial arterioles may contribute to the greater dilation of these vessels to ß2-adrenoceptor activation.
Key Words: receptors, adrenergic, beta endocardium microcirculation vasodilation
| Introduction |
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| Methods |
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RNA Isolation and Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was isolated from subepicardial and subendocardial arterioles (50- to 100-µm inner diameter, 3 to 4 vessels per sample) and from myocardial tissue (positive control), as described previously.15 Using primers specific for ß1-adrenoceptor, ß2-adrenoceptor, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, reverse transcriptionpolymerase chain reaction was conducted as described previously.15
Immunohistochemical Analysis
Subepicardial and subendocardial arterioles (50- to 100-µm inner diameter) were prepared for immunohistochemical analysis, as described previously.15 Sections (12 µm thick) were immunolabeled with a rabbit anti-ß2 antibody (1:40 dilution, Santa Cruz Biotechnology) and observed by means of confocal microscopy, as described previously.15
Data Analysis
Diameter changes in response to vasodilator agonists were normalized to the maximum diameter changes in response to 100 µmol/L sodium nitroprusside and expressed as a percentage of maximal dilation.14 Statistical comparisons were performed by means of 2-way ANOVA or Students t test, as appropriate. A value of P<0.05 was considered significant. Data are presented as mean±SEM.
| Results |
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ß-Adrenoceptor Expression in Coronary Arterioles
The reverse transcription-polymerase chain reaction results showed that ß2-adrenoceptor but not ß1-adrenoceptor mRNA expression was detected in coronary arterioles, whereas both ß-adrenoceptor subtypes were detected in myocardial tissue (Figure 2A). Normalization of ß2-adrenoceptor transcripts with GAPDH transcripts exhibited a similar level of expression for all samples evaluated and showed that the expression of ß2 was nearly 3-fold greater in subepicardial vessels than in subendocardial vessels (Figure 2A). Similarly, immunohistochemical results revealed markedly greater signal intensity for ß2-adrenoceptor protein expression in subepicardial arterioles (Figure 2B).
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| Discussion |
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Further support for a heterogeneous ß2-adrenoceptor activation was provided by procaterol, a selective ß2-adrenoceptor agonist. Similar to isoproterenol, we observed a greater dilation of subepicardial arterioles to procaterol, and the dilation of subendocardial arterioles only occurred at higher concentrations (
10 µmol/L). This is consistent with the in vivo findings that a relatively higher concentration of another ß2-adrenoceptor agonist, salbutamol (>1 µmol/L), was required to increase subendocardial blood flow in the nonworking dog hearts after ß1-adrenoceptor blockade.8 The disparity in the vasodilatory capacity to ß2-adrenoceptor agonists between subepicardial and subendocardial arterioles was apparently not a general phenomenon because the dilations of these vessels to pinacidil and sodium nitroprusside in the present study and to pinacidil and adenosine in our previous study11 were very comparable.
The pharmacological results above support the hypothesis of a heterogeneous transmural distribution of ß2-adrenoceptors. Although differences in ß2-adrenoceptor density have been determined between large and small subepicardial coronary arteries by quantitative autoradiography,6 it remains unclear whether receptor distribution, in particular at the molecular level, varies across the left ventricular wall. In agreement with the functional results, our molecular data provide the first evidence that ß2-adrenoceptor mRNA was predominantly expressed in both vessel types. However, the ß2-adrenoceptor mRNA and protein expression were significantly greater in the subepicardial vessels than in the subendocardial vessels. It appears that the differences in the sensitivity and magnitude of dilation to ß-adrenoceptor agonists between subepicardial and subendocardial arterioles may result from the unequal distribution of ß2-adrenoceptors.
Although our findings do not support a role for ß1-adrenoceptors in coronary arteriolar dilation with isoproterenol, an earlier in vivo study has shown that blockade of ß1-adrenoceptors attenuates the isoproterenol-induced coronary blood flow.16 A possible explanation for these apparently disparate results is that isoproterenol may stimulate ß1-adrenoceptors at upstream larger arterioles (>200 µm in diameter), which have been shown to contribute some degree (
25%)9 of coronary vascular resistance. Moreover, the ß1-adrenoceptors have been shown to be the predominant subtype in large coronary arteries,5 and they mediate dilation of these vessels in vitro.1 Therefore, it appears that large coronary arterioles/arteries exhibit ß1-adrenoceptors and possibly ß2-adrenoceptors, whereas small coronary arterioles, as shown in our present functional and molecular studies, exhibit predominately ß2-adrenoceptors.
Administration of the KATP channel blocker glibenclamide in the coronary circulation has been shown to attenuate ß2-adrenoceptor-induced increases in myocardial blood flow,10 suggesting that KATP channel activation contributes to the dilation of coronary resistance vessels in response to ß-adrenoceptor stimulation. This earlier study was not able to unequivocally determine whether arteriolar KATP channels are directly involved in ß-adrenoceptor-induced dilation because KATP channel activations secondary to metabolic and hemodynamic disturbances during adrenoceptor activation are inevitable in the in vivo setting. In the present study, glibenclamide inhibited the dilation of subepicardial and subendocardial arterioles to the KATP channel opener pinacidil and to isoproterenol, indicating a critical role for KATP channels in the ß2-adrenoceptorinduced dilation of these vessels. However, it does not appear that KATP channel signaling contributes to the transmural difference in vasodilation to ß-adrenoceptor agonists because pinacidil-induced dilation was similar between subepicardial and subendocardial arterioles. These results further support the idea that the differential expression of ß2-adrenoceptors rather than the downstream signaling is responsible for the observed transmural difference in coronary arteriolar dilation to ß-adrenoceptor activation.
The physiological or pathophysiological implications of the present results could provide further insight into the mechanisms contributing to regional differences in myocardial blood flow. In the normal heart, blood flow to the subendocardium is either equal to or slightly greater than that to the subepicardium. During exercise, sympathetic activation of vascular ß-adrenoceptors can contribute to coronary vasodilation3,4 and the subendocardial flow is slightly redistributed toward the subepicardium.19 For healthy individuals, this usually does not pose a grave situation, but for those with cardiovascular complications such as coronary stenosis, the redistribution of flow to the subepicardium is accentuated during exercise.20 It is worth noting that myocardial ischemia is generally associated with elevated levels of norepinephrine.21 Under this condition, the transmural difference in ß2-adrenoceptor activation, with preferential dilation in the subepicardium, could contribute, in part, to the apparent high vulnerability of subendocardium to ischemia. Indeed, in vivo studies indicate that activation of coronary ß2-adrenoceptors, independent of myocardial ß1-adrenoceptor activation, increases microvascular perfusion22 and redistributes flow toward the subepicardial region in normal7,8 and ischemic myocardium.7 Our findings may help to explain the observed differences in transmural blood flow during sympathetic activation and the greater susceptibility of subendocardium to ischemia that is exacerbated during exercise.20 A better understanding of the functional and molecular mechanisms regulating subepicardial and subendocardial tone may yield new therapies for optimizing blood flow to different regions of the heart with ischemic disease.
| Acknowledgments |
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| Footnotes |
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The online-only Data Supplement is available at http://www.circulationaha.org.
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